Dual oxidases as mitogenic and endocrine regulators

ABSTRACT

The present invention relates to new genes encoding for the production of novel proteins involved in generation of reactive oxygen intermediates and in peroxidative reactions that affect biological functions including cell division, thyroid hormone biosynthesis and tissue fibrosis. The present invention also provides vectors containing these genes, cells transfected with these vectors, antibodies raised against these novel proteins, kits for detection, localization and measurement of these genes and proteins, and methods to determine the activity of drugs to affect the activity of the proteins of the present invention.

PRIOR RELATED APPLICATIONS

This application is the National Stage of International Application No.PCT/US01/15573, filed May 14, 2001, which claims the benefit of U.S.Provisional Application No. 60/204,441, filed May 15, 2000, and U.S.Provisional Application No. 60/222,421, filed Aug. 1, 2000. Thisapplication is also a Continuation-in-Part of U.S. patent applicationSer. No. 09/437,568, filed Nov. 10, 1999 now U.S. Pat. No. 6,620,603,which claims the benefit of U.S. Provisional Application No. 60/107,911,filed Nov. 10. 1998, U.S. Provisional Application No. 60/151,242, filedAug. 27, 1999, and U.S. Provisional Application No. 60/149,332, filedAug. 17, 1999.

TECHNICAL FIELD

The present invention relates to the fields of normal and abnormal cellgrowth, in particular mitogenic regulation, to thyroid hormonebiosynthesis and to nematode cuticle biogenesis. The present inventionprovides the following: nucleotide sequences encoding for the productionof enzymes that are mitogenic regulators, that catalyze thyroid hormonebiosynthesis and in nematodes catalyze the biogenesis of cuticle; aminoacid sequences of these enzymes; vectors containing these nucleotidesequences; methods for transfecting cells with vectors that producethese enzymes; and antibodies to these enzymes that are useful fordetecting and measuring levels of these enzymes, and for binding tocells possessing extracellular epitopes of these enzymes.

BACKGROUND OF THE INVENTION

Reactive oxygen intermediates (ROI) are partial reduction products ofoxygen: 1 electron reduces O₂ to form superoxide (O₂ ⁻), and 2 electronsreduce O₂ to form hydrogen peroxide (H₂O₂). ROI are generated as abyproduct of aerobic metabolism and by toxicological mechanisms. Thereis growing evidence for regulated enzymatic generation of O₂ ⁻ and itsconversion to H₂O₂ in a variety of cells. The conversion of O₂ ⁻ to H₂O₂occurs spontaneously, but is markedly accelerated by superoxidedismutase (SOD). High levels of ROI are associated with damage tobiomolecules such as DNA, biomembranes and proteins. Recent evidenceindicates generation of ROI under normal cellular conditions and pointsto signaling and metabolic roles for O₂ ⁻ and H₂O₂.

Several biological systems generate reactive oxygen. Phagocytic cellssuch as neutrophils generate large quantities of ROI as part of theirbattery of bactericidal mechanisms. Exposure of neutrophils to bacteriaor to various soluble mediators such as formyl-Met-Leu-Phe or phorbolesters activates a massive consumption of oxygen, termed the respiratoryburst, to initially generate superoxide, with secondary generation ofH₂O₂, HOCl and hydroxyl radical. The enzyme responsible for this oxygenconsumption is the respiratory burst oxidase (nicotinamide adeninedinucleotide phosphate-reduced form (NADPH) oxidase).

There is growing evidence for the generation of ROI by non-phagocyticcells, particularly in situations related to cell proliferation. Inaddition in some tissues such as thyroid, ROI generation is implicatedin metabolic conversions such as the biosynthesis of thyroid hormone.Significant generation of H₂O₂, O₂ ⁻, or both have been noted in somecell types. Fibroblasts and human endothelial cells show increasedrelease of superoxide in response to cytokines such as interleukin-1 ortumor necrosis factor (TNF) (Meier et al. (1989) Biochem J. 263,539–545.; Matsubara et al. (1986) J. Immun. 137, 3295–3298).Ras-transformed fibroblasts show increased superoxide release comparedwith control fibroblasts (Irani, et al. (1997) Science 275, 1649–1652).Rat vascular smooth muscle cells show increased H₂O₂ release in responseto PDGF (Sundaresan et al. (1995) Science 270, 296–299) and angiotensinII (Griendling et al. (1994) Circ. Res. 74, 1141–1148; Fukui et al.(1997) Circ. Res. 80, 45–51; Ushio-Fukai et al. (1996) J. Biol. Chem.271, 23317–23321), and H₂O₂ in these cells is associated with increasedproliferation rate. The occurrence of ROI in a variety of cell types issummarized in Table 1 (adapted from Burdon, R. (1995) Free Radical Biol.Med. 18, 775–794).

TABLE 1 Superoxide Hydrogen Peroxide human fibroblasts Balb/3T3 cellshuman endothelial cells rat pancreatic isletcells human/rat smoothmuscle cells murine keratinocytes human fat cells rabbit chondrocyteshuman osteocytes human tumor cells BHK-21 cells fat cells, 3T3 L1 cellshuman colonic epithelial cells

ROI generated by the neutrophil have a cytotoxic function. While ROI arenormally directed at the invading microbe, ROI can also induce tissuedamage (e.g., in inflammatory conditions such as arthritis, shock, lungdisease, and inflammatory bowel disease) or may be involved in tumorinitiation or promotion, due to damaging effects on DNA. Nathan(Szatrowski et al. (1991) Canc. Res. 51, 794–798) proposed that thegeneration of ROI in tumor cells may contribute to the hypermutabilityseen in tumors, and may therefore contribute to tumor heterogeneity,invasion and metastasis.

In addition to cytotoxic and mutagenic roles, ROI have ideal propertiesas signal molecules: 1) they are generated in a controlled manner inresponse to upstream signals; 2) the signal can be terminated by rapidmetabolism of O₂ ⁻ and H₂O₂ by SOD and catalase/peroxidases; 3) theyelicit downstream effects on target molecules, e.g., redox-sensitiveregulatory proteins such as NF kappa B and AP-1 (Schreck et al. (1991)EMBO J. 10, 2247–2258; Schmidt et al. (1995) Chemistry & Biology 2,13–22). Oxidants such as O₂ ⁻ and H₂O₂ have a relatively well definedsignaling role in bacteria, operating via the SoxI/II regulon toregulate transcription.

ROI appear to have a direct role in regulating cell division, and mayfunction as mitogenic signals in pathological conditions related togrowth. These conditions include cancer and cardiovascular disease. O₂ ⁻is generated in endothelial cells in response to cytokines, and mightplay a role in angiogenesis (Matsubara et al. (1986) J. Immun.137,3295–3298). O₂ ⁻ and H₂O₂ are also proposed to function as“life-signals”, preventing cells from undergoing apoptosis (Matsubara etal. (1986) J. Immun. 137, 3295–3298). As discussed above, many cellsrespond to growth factors (e.g., platelet derived growth factor (PDGF),epidermal derived growth factor (EGF), angiotensin II, and variouscytokines) with both increased production of O₂ ⁻/H₂O₂ and increasedproliferation. Inhibition of ROI generation prevents the mitogenicresponse. Exposure to exogenously generated O₂ ⁻ and H₂O₂ results in anincrease in cell proliferation. A partial list of responsive cell typesis shown below in Table 2 (adapted from Burdon, R. (1995) Free RadicalBiol. Med. 18, 775–794).

TABLE 2 Superoxide Hydrogen peroxide human, hamster fibroblasts mouseosteoblastic cells Balb/3T3 cells Balb/3T3 cells human histiocyticleukemia rat, hamster fibroblasts mouse epidermal cells human smoothmuscle cells rat colonic epithelial cells rat vascular smooth muscle ratvascular smooth muscle cells cells

While non-transformed cells can respond to growth factors and cytokineswith the production of ROI, tumor cells appear to produce ROI in anuncontrolled manner. A series of human tumor cells produced largeamounts of hydrogen peroxide compared with non-tumor cells (Szatrowskiet al. (1991) Canc. Res. 51, 794–798). Ras-transformed NIH 3T3 cellsgenerated elevated amounts of superoxide, and inhibition of superoxidegeneration by several mechanisms resulted in a reversion to a “normal”growth phenotype.

O₂ ⁻ has been implicated in maintenance of the transformed phenotype incancer cells including melanoma, breast carcinoma, fibrosarcoma, andvirally transformed tumor cells. Decreased levels of the manganese formof SOD (MnSOD) have been measured in cancer cells and invitro-transformed cell lines, predicting increased O₂ ⁻ levels (Burdon,R. (1995) Free Radical Biol. Med. 18, 775–794). MnSOD is encoded onchromosome 6q25 which is very often lost in melanoma. Overexpression ofMnSOD in melanoma and other cancer cells (Church et al. (1993) Proc. ofNatl. Acad. Sci. 90, 3113–3117; Fernandez-Pol et al. (1982) Canc. Res.42, 609–617; Yan et al. (1996) Canc. Res. 56, 2864–2871) resulted insuppression of the transformed phenotype.

ROI are implicated in growth of vascular smooth muscle associated withhypertension, atherosclerosis, and restenosis after angioplasty. O₂ ⁻generation is seen in rabbit aortic adventitia (Pagano et al. (1997)Proc. Natl. Acad. Sci. 94, 14483–14488). Vascular endothelial cellsrelease O₂ ⁻ in response to cytokines (Matsubara et al. (1986) J. Immun.137, 3295–3298). O₂ ⁻ is generated by aortic smooth muscle cells inculture, and increased O₂ ⁻ generation is stimulated by angiotensin IIwhich also induces cell hypertrophy. In a rat model system, infusion ofangiotensin II leads to hypertension as well as increased O₂ ⁻generation in subsequently isolated aortic tissue (Ushio-Fukai et al.(1996) J. Biol. Chem. 271, 23317–23321.; Yu et al. (1997) J. Biol. Chem.272, 27288–27294). Intravenous infusion of a form of SOD that localizesto the vasculature or an infusion of an O₂ ⁻ scavenger preventedangiotensin II induced hypertension and inhibited ROI generation (Fukuiet al. (1997) Circ. Res. 80, 45–51).

The neutrophil NADPH oxidase, also known as phagocyte respiratory burstoxidase, provides a paradigm for the study of the specialized enzymaticROI-generating system. This extensively studied enzyme oxidizes NADPHand reduces oxygen to form O₂ ⁻. NADPH oxidase consists of multipleproteins and is regulated by assembly of cytosolic and membranecomponents. The catalytic moiety consists of flavocytochrome b₅₅₈, anintegral plasma membrane enzyme comprised of two components: gp91phox(gp refers to glycoprotein; phox is an abbreviation of the wordsphagocyte and oxidase) and p22phox (p refers to protein). gp91phoxcontains 1 flavin adenine dinucleotide (FAD) and 2 hemes as well as theNADPH binding site.

p22phox has a C-terminal proline-rich sequence which serves as a bindingsite for cytosolic regulatory proteins. The two cytochrome subunits,gp91phox and p22phox appear to stabilize one another, since the geneticabsence of either subunit, as in the inherited disorder chronicgranulomatous disease (CGD), results in the absence of the partnersubunit (Yu et al. (1997) J. Biol. Chem. 272. 27288–27294). Essentialcytosolic proteins include p47phox, p67phox and the small GTPase Rac, ofwhich there are two isoforms. p47phox and p67phox both contain SH₃regions and proline-rich regions which participate in proteininteractions governing assembly of the oxidase components duringactivation. The neutrophil enzyme is regulated in response to bacterialphagocytosis or chemotactic signals by phosphorylation of p47phox, andperhaps other components, as well as by guanine nucleotide exchange toactivate the GTP-binding protein Rac.

The origin of ROI in non-phagocytic tissues is unproven, but theoccurrence of phagocyte oxidase components has been evaluated in severalsystems by immunochemical methods, Northern blots and reversetranscriptase-polymerase chain reaction (RT-PCR). The message forp22phox is expressed widely, as is that for Rac1. Several cell typesthat are capable of O₂ ⁻ generation have been demonstrated to containall of the phox components including gp91phox, as summarized below inTable 3. These cell types include endothelial cells, aortic adventitiaand lymphocytes.

TABLE 3 Tissue gp91phox p22phox p47phox p67phox neutrophil +^(1,2)+^(1,2) +^(1,2) +^(1,2) aortic adventitia +¹ +¹ +¹ +¹ lymphocytes +² +²+^(1,2) +^(1,2) endothelial cells +² +² +^(1,2) +^(1,2) glomerularmesangial — +^(1,2) +^(1,2) +^(1,2) cells fibroblasts — +² +^(1,2) +²aortic sm. muscle — +^(1,2) ? ? ¹= protein expression shown. ²= mRNAexpression shown.

A distinctly different pattern is seen in several other cell types shownin Table 3 including glomerular mesangial cells, rat aortic smoothmuscle and fibroblasts. In these cells, expression of gp91phox is absentwhile p22phox and in some cases cytosolic phox components have beendemonstrated to be present. Since gp91phox and p22phox stabilize oneanother in the neutrophil, there has been much speculation that somemolecule, possibly related to gp91phox, accounts for ROI generation inglomerular mesangial cells, rat aortic smooth muscle and fibroblasts(Ushio-Fukai et al. (1996) J. Biol. Chem. 271, 23317–23321).Investigation of fibroblasts from a patient with a genetic absence ofgp91phox provides proof that the gp91phox subunit is not involved in ROIgeneration in these cells (Emmendorffer et al. (1993) Eur. J. Haematol.51, 223–227). Depletion of p22phox from vascular smooth muscle using anantisense approach indicated that this subunit participates in ROIgeneration in these cells, despite the absence of detectable gp91phox(Ushio-Fukai et al. (1996) J. Biol. Chem. 271, 23317–23321).

Thyroid hormone regulates basal metabolic rate through end-effects onmitochondrial respiration, and conditions of under- or over-productionare important clinically. Development of drugs to regulate thebiosynthesis of thyroid hormone is a medically important goal, andidentification of the enzymes in this pathway is key to developingpharmacologically relevant targets. Thyroid uniquely concentratesiodide, which is used to iodinate tyrosine residues on thyroglobulin(TG). TG is a large protein (660 kDa) that contains 67 tyrosyl residues,some of which are preferential sites for iodination. Iodination oftyrosines in TG is catalyzed by thyroid peroxidase (TPO), a plasmamembrane hemoprotein. Iodination requires a previously unidentifiedenzymatic source of H₂O₂. A second step is the coupling of twodiiodotyrosines (DIT) to form protein-associated thyroxine (T4), whichis subsequently proteolytically cleaved from TG to liberate free T4.What is needed is a composition of the gene that encodes the enzyme thatgenerates H₂O₂ in thyroid and that catalyzes the coupling reaction, anda method of using that composition to modulate thyroid hormonebiosynthesis. Such information would be useful in the development ofdrugs for modulation of thyroid function. Such modulation might beuseful in the treatment of hyperthyroidism.

Recent evidence suggests that enzymes involved in oxidativecross-linking of tyrosine in growth factor stimulated fibroblasts maylead to fibrotic damage. Lung fibrosis is particularly damaging toindividuals afflicted with this condition. Identification of the genesencoding enzymes involved in such oxidative cross-linking reactions isneeded so that drugs may be designed to alleviate or prevent fibroticdamage, particularly in the lung.

Parasitic diseases are a major cause of morbidity and mortalityworldwide in humans and animals, and have a significant impact onagricultural productivity as well. Parasitic diseases have provendifficult to treat, in part due to the presence of the cuticle, a toughexoskeletal structure of parasites such as nematodes. What is needed isa composition and method of using the composition to fight parasiticdiseases, including but not limited to those parasitic diseases causedby parasites with cuticles.

Accordingly, what is needed is a method of disrupting the formation ofthe cuticle which would make the worm susceptible to the host defensemechanisms and drug treatment.

What is also needed is the identity of the proteins involved in ROIgeneration, especially in non-phagocytic tissues and cells. What is alsoneeded are the nucleotide sequences encoding for these proteins, and theprimary sequences of the proteins themselves. Also needed are vectorsdesigned to include nucleotides encoding for these proteins. Probes andPCR primers derived from the nucleotide sequence are needed to detect,localize and measure nucleotide sequences, including mRNA, involved inthe synthesis of these proteins. In addition, what is needed is a meansto transfect cells with these vectors. What is also needed areexpression systems for production of these molecules. Also needed areantibodies directed against these molecules for a variety of usesincluding localization, detection, and measurement and passiveimmunization.

SUMMARY OF THE INVENTION

The present invention solves the problems described above by providing anovel family of nucleotide sequences and proteins encoded by thesenucleotide sequences termed duox proteins. In particular, the presentinvention provides compositions comprising the nucleotide sequences SEQID NO: 1 and SEQ ID NO: 3, and fragments and conservative substitutionsthereof, which encode for the expression of proteins comprising SEQ IDNO: 2 and SEQ ID NO: 4 respectively, and fragments and conservativesubstitutions thereof Preferred protein fragments include, but are notlimited to, SEQ ID NO: 31 and SEQ ID NO: 32. While not wanting to bebound by the following statement, it is believed that these proteins areinvolved in ROI production and are capable of stimulating superoxideproduction or generating peroxidative reactions. The present inventionalso provides vectors containing these nucleotide sequences, cellstransfected with these vectors which produce the proteins comprising SEQID NO: 2 and SEQ ID NO: 4, and fragments and conservative substitutionsthereof, and antibodies to these proteins and fragments and conservativesubstitutions thereof The present invention also provides methods forstimulating cellular proliferation by administering vectors encoded forproduction of the proteins comprising SEQ ID NO: 2 and SEQ ID NO: 4 andfragments and conservative substitutions thereof The present inventionalso provides methods for stimulating cellular proliferation byadministering the proteins comprising SEQ ID NO: 2 and SEQ ID NO: 4 andfragments and conservative substitutions thereof. The proteinscomprising SEQ ID NO: 2 and SEQ ID NO: 4 and fragments and conservativesubstitutions thereof are useful in affecting the exoskeleton,especially the cuticle of parasites, including but not limited tonematodes. The nucleotides and antibodies of the present invention areuseful for the detection, localization and measurement of the nucleicacids encoding for the production of the proteins of the presentinvention, and also for the detection, localization and measurement ofthe proteins of the present invention. These nucleotides and antibodiescan be combined with other reagents in kits for the purposes ofdetection, localization and measurement.

Most particularly, the present invention involves a method forregulation of cell division or cell proliferation by modifying theactivity or expression of the duox proteins described as SEQ ID NO: 2and SEQ ID NO: 4 or fragments or conservative substitutions thereof.These proteins, in their naturally occurring or expressed forms, areexpected to be useful in drug development, for example for screening ofchemical and drug libraries by observing inhibition of the activity ofthese enzymes. Such chemicals and drugs would likely be useful astreatments for cancer, prostatic hypertrophy, benign prostatichypertrophy, hypertension, metabolic disease, fibrosis, atherosclerosisand many other disorders involving abnormal cell growth orproliferation, and a variety of parasitic diseases in both animals andcrops as described below. The entire expressed protein may be useful inthese assays. Portions of the molecule which may be targets forinhibition or modification include but are not limited to the bindingsite for pyridine nucleotides (NADPH or NADH), the flavoprotein domain(approximately the C-terminal 265 amino acids), and/or the binding orcatalytic site for flavin adenine dinucleotide (FAD).

The method of the present invention may be used for the development ofdrugs or other therapies for the treatment of conditions associated withabnormal growth including, but not limited to the following: cancer,fibrosis, lung fibrosis, metabolic imbalances, thyroid imbalances,hyperthryoidism, psoriasis, prostatic hypertrophy, benign prostatichypertrophy, cardiovascular disease, proliferation of vessels, includingbut not limited to blood vessels and lymphatic vessels, arteriovenousmalformation, vascular problems associated with the eye,atherosclerosis, hypertension, and restenosis following angioplasty andparasitic diseases. The enzymes of the present invention are excellenttargets for the development of drugs and other agents which may modulatethe activity of these enzymes. It is to be understood that modulation ofactivity may result in enhanced, diminished or absence of enzymaticactivity. Modulation of the activity of these enzymes may be useful intreatment of conditions, including but not limited to conditionsassociated with abnormal growth, metabolic disorders, and fibrosis.

Drugs which affect the activity of the duox enzymes represented in SEQID NO: 2 and SEQ ID NO: 4, or fragments or conservative substitutionsthereof, may also be combined with other therapeutics in the treatmentof specific conditions. For example, these drugs may be combined withangiogenesis inhibitors in the treatment of cancer, withantihypertensives for the treatment of hypertension, with cholesterollowering drugs for the treatment of atherosclerosis and with hormonalagonist or antagonists in the treatment of endocrine disorders, such asthyroid disorders.

It is to be understood that the proteins of the present invention,including but not limited to, SEQ ID NO: 2 and SEQ ID NO: 4, orfragments or conservative substitutions thereof, may be administeredtogether with other compositions such as anti-parasitic compositions,pesticides, herbicides and fertilizers. Accordingly, the proteins of thepresent invention may be useful alone or in combination with othercompositions for treating humans or animals, including livestock, otherfarm animals and domestic animals, including pets, for preventing orfighting parasitic disease, for protecting plants and crops againstattack by parasites, especially soil nematodes, and for destroyingparasites.

Accordingly, an object of the present invention is to provide nucleotidesequences, or fragments thereof or conservative substitutions thereof,encoding for the production of proteins, or fragments thereof orconservative substitutions thereof, that are involved in ROI production,stimulate superoxide production or generate peroxidative reactions.

It is another object of the present invention is to provide the proteinsrepresented in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 31, and SEQ ID NO:32 or fragments Or conservative substitutions thereof.

It is another object of the present invention is to provide thenucleotide sequences encoding for the proteins represented in SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 31, and SEQ ID NO: 32 or fragments orconservative substitutions thereof, wherein these nucleotide sequenceinclude SEQ ID NO: 1, SEQ ID NO: 3 or fragments or conservativesubstitutions thereof.

It is another object of the present invention to provide proteins,fragments thereof or conservative substitutions thereof, involved inexoskeletal or cuticle formation that may be used as targets fortherapies designed to prevent exoskeletal or cuticle formation and toharm organisms having an exoskeleton or cuticle, particularly parasites.

It is another object of the present invention to provide proteins,fragments thereof or conservative substitutions thereof, involved inthyroid hormone biosynthesis that may be used as targets for therapiesdesigned to inhibit biosynthesis of thyroid hormone.

It is yet another object of the present invention to provide proteins,fragments thereof or conservative substitutions thereof, involved intissue fibrosis that may be used as targets for therapies designed toprevent fibrosis.

Another object of the present invention is to provide proteins involvedin lung fibrosis that may be used as targets for therapies designed toprevent lung fibrosis.

Another object of the present invention is to provide vectors containingthese nucleotide sequences, or fragments thereof.

Yet another object of the present invention is to provide cellstransfected with these vectors.

Still another object of the present invention is to administer cellstransfected with these vectors to animals and humans.

Another object of the present invention is to provide proteins, orfragments thereof, that are involved in ROI production, stimulatesuperoxide production or generate peroxidative reactions.

Still another object of the present invention is to provide antibodies,including monoclonal and polyclonal antibodies, or fragments thereof,raised against proteins, or fragments thereof or conservativesubstitutions thereof, that are involved in ROI production, stimulatesuperoxide production or generate peroxidative reactions. Suchantibodies are useful in the localization and measurement of proteins,or fragments thereof, that are involved in ROI production.

Another object of the present invention is to administer genescontaining nucleotide sequences, or fragments thereof, encoding for theproduction of proteins, or fragments thereof or conservativesubstitutions thereof, that are involved in ROI production, stimulatesuperoxide production or generate peroxidative reactions, to animals andhumans and also to cells obtained from animals and humans.

Another object of the present invention is to administer antisensecomplimentary sequences of genes containing nucleotide sequences, orfragments thereof or conservative substitutions thereof, encoding forthe production of proteins, or fragments thereof or conservativesubstitutions thereof, that are involved in ROI production, stimulatesuperoxide production or generate peroxidative reactions, to animals andhumans and also to cells obtained from animals and humans.

Yet another object of the present invention is to provide a method forstimulating or inhibiting cellular proliferation by administeringvectors containing nucleotide sequences, or fragments thereof, encodingfor the production of proteins, or fragments thereof, that are involvedin ROI production, stimulate superoxide production or generateperoxidative reactions, to animals and humans. It is also an object ofthe present invention to provide a method for stimulating or inhibitingcellular proliferation by administering vectors containing antisensecomplimentary sequences of nucleotide sequences, or fragments thereof,encoding for the production of proteins, or fragments thereof, that areinvolved in ROI production, stimulate superoxide production or generateperoxidative reactions, to animals and humans. These methods ofstimulating cellular proliferation are useful for a variety of purposes,including but not limited to, developing animal models of tumorformation, stimulating cellular proliferation of blood marrow cellsfollowing chemotherapy or radiation, or in cases of anemia.

Yet another object of the present invention is to provide nucleotideprobes useful for the detection, localization and measurement ofnucleotide sequences, or fragments thereof, encoding for the productionof proteins, or fragments thereof, that are involved in ROI production,stimulate superoxide production or generate peroxidative reactions.

Another object of the present invention is to provide kits useful fordetection of nucleic acids including the nucleic acids represented inSEQ ID NO: 1, and SEQ ID NO: 3, or fragments thereof or conservativesubstitutions thereof, that encode for proteins, or fragments thereof orconservative substitutions thereof, that are involved in ROI production,stimulate superoxide production or generate peroxidative reactions.

Yet another object of the present invention is to provide kits usefulfor detection and measurement of nucleic acids including the nucleicacids represented in SEQ ID NO: 1, and SEQ ID NO: 3, or fragmentsthereof, that encode for proteins, or fragments thereof or conservativesubstitutions thereof, that are involved in ROI production, stimulatesuperoxide production or generate peroxidative reactions.

Another object of the present invention is to provide kits useful fordetection of proteins, including the proteins represented in SEQ ID NO:2 and SEQ ID NO: 4 or fragments thereof, that are involved in ROIproduction, stimulate superoxide production or generate peroxidativereactions.

Yet another object of the present invention is to provide kits usefulfor detection and measurement of proteins, including the proteinsrepresented in SEQ ID NO: 2 and SEQ ID NO: 4, or fragments thereof, thatare involved in ROI production, stimulate superoxide production orgenerate peroxidative reactions.

Still another object of the present invention is to provide kits usefulfor localization of proteins, including the proteins represented in SEQID NO: 2 and SEQ ID NO: 4, or fragments thereof, that are involved inROI production, stimulate superoxide production or generate peroxidativereactions.

Yet another object of the present invention is to provides kits usefulfor the detection, measurement or localization of nucleic acids, orfragments thereof, encoding for proteins, or fragments thereof, that areinvolved in ROI production, for use in diagnosis and prognosis ofabnormal cellular proliferation related to ROI production.

Another object of the present invention is to provides kits useful forthe detection, measurement or localization of proteins, or fragmentsthereof, that are involved in ROI production, for use in diagnosis andprognosis of abnormal cellular proliferation related to ROI production.

These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiments and the appended drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Structure and sequence alignments of large homologs of gp9phox.Sequence alignment of the first and second EF hand domains from h-doux(SEQ ID NO: 2, residues 823 to 851 and 859 to 887 respectively), thefirst and second EF hand domains from Ce-doux1, (SEQ ID NO: 3, residues821 to 849 and 857 to 885 respectively), and the first and second EFhand domains from Ce-doux2 (SEQ ID NO: 3 residues 821 to 849 and 857 to885 respectively). Domain structure of Duox proteins. Secretory signalpeptide sequences are indicated by a gray triangle, while predictedtransmembrane alpha helices are indicated by a hashed rectangle. Whiteovals indicate regions showing homology with EF-hand calcium bindingsites.

FIG. 2. Comparison of the peroxidase domains of h-Duox, Ce-Duox1 andsome known peroxidases. A) Sequence alignments of human MPO (SEQ ID NO:33), human TPO (SEQ ID NO: 34), human EPO (SEQ ID NO: 35), bovine LPO(residues 61 to 620 of SEQ ID NO: 36). Drosophila Pxsn-dors (SEQ ID NO:37), human Doux1 (SEQ ID NO: 38), human Doux2 (residues 40–574 of SEQ IDNO: 31) and C. elegans Ce-Doux1 (residues 33–566 of SEQ ID NO: 32).Abbreviations are: MPO, myeloperoxidase; TPO, thyroid peroxidase; EPO,eosinophil peroxidase; LPO, lactoperoxidase, Pxsn.dros, Drosophilaperoxidasin. Residues which are conserved among all 7 proteins are shownwith black boxes, while those matching a derived consensus sequence areshown in line boxes. Filled circles indicate residues which are proposedto provide contacts with the heme, based on the crystal structure ofcanine myeloperoxidase (Zeng and Feima, 1992). The superscripted doublebar indicates residues comprising a calcium binding region, and filledtriangles indicate residues which appear in the crystal structure tobind directly to the calcium ion.

FIG. 3. Tissue expression of mRNA for h-Duox. mRNA for h-Duox1, h-Duox2and glyceraldehyde 3-phosphate dehydrogenase was detected by RT-PCR.

DETAILED DESCRIPTION OF THE INVENTION

The present invention solves the problems described above by providing anovel family of nucleotide sequences and proteins, encoded by thesenucleotide sequences, termed duox proteins. The term “duox” refers to“dual oxidase”. In particular, the present invention provides novelcompositions comprising the nucleotide sequences SEQ ID NO: 1, and SEQID NO: 3, and fragments thereof or conservative substitutions thereof,which encode, respectively, for the expression of proteins comprisingSEQ ID NO: 2 and SEQ ID NO: 4 and fragments thereof or conservativesubstitutions thereof. Preferred protein fragments include, but are notlimited to SEQ ID NO: 31 and SEQ ID NO: 32.

The duox proteins described herein have homology to the gp91phox proteininvolved in ROI generation, however, the duox proteins comprise a noveland distinct family of proteins. The duox proteins described herein havethree distinct regions: the amino terminal region having homology toperoxidase proteins, the internal region having homology to calmodulin(CAM) proteins and the carboxy-terminal region having homology to mox(also called nox) proteins. The amino acid sequence of human duox2 isshown in SEQ ID NO: 2. Nucleotides encoding duox2 proteins are alsoshown in SEQ ID NO: 1. In addition to the human duox proteins,comparison of the sequence of human duox1 and human duox2 with genomicdatabases using BLAST searching resulted in the identification of twohomologs of duox in C. elegans (Ce-duox1 SEQ ID NO: 3) and thepseudogene Ce-duox2. Drosophila also appears to have at least one duoxhomolog. Thus, the duox family of genes/proteins is widely distributedamong multicellular organisms.

High molecular weight homologs of gp91phox, have been identified inhuman (h) and C. elegans (Ce), and are termed Duox for “dual oxidase”because they have both a peroxidase-homology domain and a gp91phoxdomain. Ce-Duox uses cytosolic NADPH to generate reactive oxygen. Itcatalyzes cross-linking of free tyrosine ethyl ester involved in thestabilization of the cuticular extracellular matrix in nematodes.

Although not wanting to be bound by the following statement, it isbelieved that duox enzymes, for example duox2 and Ce-duox1, have dualenzymatic functions, catalyzing both the generation of superoxide andperoxidative type reactions. The latter class of reactions utilizehydrogen peroxide as a substrate (and in some cases have been proposedto utilize superoxide as a substrate). Since hydrogen peroxide isgenerated spontaneously from the dismutation of superoxide, it isbelieved that the NAD(P)H oxidase domain generates the superoxide and/orhydrogen peroxide which can then be used as a substrate for theperoxidase domain. In support of this hypothesis, a model for the duox2protein in C. elegans has been developed that has an extracellularN-terminal peroxidase domain, a transmembrane region and a NADPH bindingsite located on the cytosolic face of the plasma membrane. By analogywith the neutrophil NADPH-oxidase which generates extracellularsuperoxide, human duox2 is predicted to generate superoxide and itsbyproduct hydrogen peroxide extracellularly where it can be utilized bythe peroxidase domain.

The peroxidase domain is likely to confer additional biologicalfunctions. Depending upon the co-substrate, peroxidases can participatein a variety of reactions including halogenation such as the generationof hypochlorous acid (HOCl) by myeloperoxidase and the iodination oftyrosine to form thyroxin by thyroid peroxidase. Peroxidases have alsobeen documented to participate in the metabolism of polyunsaturatedfatty acids, and in the chemical modification of tyrosine in collagen(by sea urchin ovoperoxidase). Although not wanting to be bound by thisstatement, it is believed that the predicted transmembrane nature ofduox2 facilitates its function in the formation or modification ofextracellular matrix or basement membrane. Since the extracellularmatrix plays an important role in tumor cell growth, invasion andmetastasis, it is believed that the duox type enzymes play a pathogenicrole in such conditions.

In addition to the nucleotide sequences described above, the presentinvention also provides vectors containing these nucleotide sequencesand fragments thereof or conservative substitutions thereof, cellstransfected with these vectors which produce the proteins comprising SEQID NO: 2 and SEQ ID NO: 4 and fragments thereof or conservativesubstitutions thereof, and antibodies to these proteins and fragmentsthereof. The present invention also provides methods for stimulatingcellular proliferation by administering vectors, or cells containingvectors, encoded for production of the proteins comprising SEQ ID NO: 2,SEQ ID NO: 4, and fragments thereof. The nucleotides and antibodies ofthe present invention are useful for the detection, localization andmeasurement of the nucleic acids encoding for the production of theproteins of the present invention, and also for the detection,localization and measurement of the proteins of the present invention.These nucleotides and antibodies can be combined with other reagents inkits for the purposes of detection, localization and measurement. Thesekits are useful for diagnosis and prognosis of conditions involvingcellular proliferation associated with production of reactive oxygenintermediates.

The present invention solves the problems described above by providing acomposition comprising the nucleotide sequence SEQ ID NO: 1 andfragments thereof and conservative substitutions thereof. The presentinvention also provides a composition comprising the nucleotide sequenceSEQ ID NO: 3 and fragments thereof and conservative substitutionsthereof. The present invention provides a composition comprising theprotein SEQ ID NO: 2, and fragments and conservative substitutionsthereof, encoded by the nucleotide sequence SEQ ID NO: 1 and fragmentsand conservative substitutions thereof The present invention provides acomposition comprising the protein SEQ ID NO: 4 and fragments andconservative substitutions thereof, encoded by the nucleotide sequenceSEQ ID NO: 3 and fragments and conservative substitutions thereof.Preferred protein fragments include, but are not limited to, SEQ ID NO:31 and SEQ ID NO: 32.

The present invention also provides vectors containing the nucleotidesequences SEQ ID NO: 1, and SEQ ID NO: 3 or fragments thereof Thepresent invention also provides cells transfected with these vectors.

In addition, the present invention provides cells stably transfectedwith the nucleotide sequence SEQ ID NO: 1 or fragments thereof. Thepresent invention also provides cells stably transfected with thenucleotide sequence SEQ ID NO: 3 or fragments thereof.

The present invention provides cells stably transfected with thenucleotide sequence SEQ ID NO: 1 or fragments or conservativesubstitutions thereof, which produce the protein SEQ ID NO: 2 orfragments or conservative substitutions thereof. In addition, thepresent invention provides cells stably transfected with the nucleotidesequence SEQ ID NO: 3 or fragments or conservative substitutions thereofwhich produce the protein SEQ ID NO: 4 or fragments or conservativesubstitutions thereof.

The present invention provides a method for stimulating growth byadministering cells stably transfected with the nucleotide sequence SEQID NO: 1 or fragments or conservative substitutions thereof whichproduce the protein SEQ ID NO: 2 or fragments or conservativesubstitutions thereof. The present invention also provides a method forstimulating growth by administering cells stably transfected with thenucleotide sequence SEQ ID NO: 3 or fragments or conservativesubstitutions thereof, which produce the protein SEQ ID NO: 4 orfragments or conservative substitutions thereof.

Specifically, the present invention provides a method for stimulatingtumor formation by administering cells stably transfected with thenucleotide sequence SEQ ID NO: 1 or fragments thereof, which produce theprotein SEQ ID NO: 2 or fragments thereof. The present invention alsoprovides a method for stimulating tumor formation by administering cellsstably transfected with the nucleotide sequence SEQ ID NO: 3 orfragments thereof, which produce the protein SEQ ID NO: 4 or fragmentsthereof. The present invention may also be used to develop anti-sensenucleotide sequences to SEQ ID NO: 1 and SEQ ID NO: 3, or fragmentsthereof. These anti-sense molecules may be used to interfere withtranslation of nucleotide sequences, such as SEQ ID NO: 1, and SEQ IDNO: 3, or fragments thereof, that encode for proteins such as SEQ ID NO:2, SEQ ID NO: 4, or fragments thereof. Administration of theseanti-sense molecules, or vectors encoding for anti sense molecules, tohumans and animals, would interfere with production of proteins such asSEQ ID NO: 2, SEQ ID NO: 4, or fragments thereof, thereby decreasingproduction of ROIs and inhibiting cellular proliferation. These methodsare useful in producing animal models for use in study of tumordevelopment, cuticle formation and vascular growth, and for study of theefficacy of treatments for affecting tumor growth, vascular growth andcuticle formation in vivo.

The present invention also provides a method for high throughputscreening of drugs and chemicals which modulate the proliferativeactivity of the enzymes of the present invention or fragments orconservative substitutions thereof, thereby affecting cell division,metabolic activity, cuticle formation, fibrosis and other biologicalfunctions involving oxidative reactions. Combinatorial chemicallibraries may be screened for chemicals which modulate the proliferativeactivity or oxidative activity of these enzymes. Drugs and chemicals maybe evaluated based on their ability to modulate the enzymatic activityof the expressed or endogenous proteins, including those represented SEQID NO: 2 and SEQ ID NO: 4 or fragments or conservative substitutionsthereof. Endogenous proteins may be obtained from many different tissuesor cells, such as colon cells. Drugs may also be evaluated based ontheir ability to bind to the expressed or endogenous proteinsrepresented by SEQ ID NO: 2 and SEQ ID NO: 4 or fragments orconservative substitutions thereof Enzymatic activity may be NADPH- orNADH-dependent superoxide generation catalyzed by the holoprotein.Enzymatic activity may also be NADPH- or NADH-dependent diaphoraseactivity catalyzed by either the holoprotein or the flavoprotein domain.

By flavoprotein domain, is meant approximately the C-terminal half ofthe enzymes shown in SEQ ID NO: 2 and SEQ ID NO: 4, or fragments orconservative substitutions thereof, (approximately the C-terminal 265amino acids). This fragment of gp91phox has NADPH-dependent reductaseactivity towards cytochrome c, nitrobluetetrazolium and other dyes.Expressed proteins or fragments thereof can be used for robotic screensof existing combinatorial chemical libraries. While not wanting to bebound by the following statement, it is believed that the NADPH or NADHbinding site and the FAD binding site are useful for evaluating theability of drugs and other compositions to bind to the duox enzymes orfragments or conservative substitutions thereof, or to modulate theirenzymatic activity. The use of the holoprotein or the C-terminal half orend regions are preferred for developing a high throughput drug screen.Additionally, the N-terminal one-third of the duox domain (theperoxidase domain) may also be used to evaluate the ability of drugs andother compositions to inhibit the peroxidase activity, and for furtherdevelopment of a high throughput drug screen.

The present invention also provides antibodies directed to the oxidativeenzymes such as SEQ ID NO: 2 and SEQ ID NO: 4 and fragments orconservative substitutions thereof. Preferred protein fragments include,but are not limited to, SEQ ID NO: 31 and SEQ ID NO: 32. The antibodiesof the present invention are useful for a variety of purposes includinglocalization, detection and measurement of the proteins SEQ ID NO: 2 andSEQ ID NO: 4 and fragments or conservative substitutions thereof. Theantibodies may be employed in kits to accomplish these purposes. Theseantibodies may also be linked to cytotoxic agents for selected killingof cells. The term antibody is meant to include any class of antibodysuch as IgG, IgM and other classes. The term antibody also includes acompletely intact antibody and also fragments thereof, including but notlimited to Fab fragments and Fab+Fc fragments.

The present invention also provides the nucleotide sequences SEQ ID NO:1 and SEQ ID NO:. 3 and fragments or conservative substitutions thereof.These nucleotides are useful for a variety of purposes includinglocalization, detection, and measurement of messenger RNA involved insynthesis of the proteins represented as SEQ ID NO: 2 and SEQ ID NO: 4and fragments or conservative substitutions thereof. These nucleotidesmay also be used in the construction of labeled probes for thelocalization, detection, and measurement of nucleic acids such asmessenger RNA or alternatively for the isolation of larger nucleotidesequences containing the nucleotide sequences shown in SEQ ID NO: 1, andSEQ ID NO: 3 or fragments or conservative substitutions thereof. Thesenucleotide sequences may be used to isolate homologous strands fromother species using techniques known to one of ordinary skill in theart. These nucleotide sequences may also be used to make probes andcomplementary strands.

Most particularly, the present invention involves a method formodulation of growth by modifying the proteins represented as SEQ ID NO:2 and SEQ ID NO: 4 or fragments or conservative substitutions thereof.

The term “mitogenic regulators” is used herein to mean any molecule thatacts to affect cell division.

The term “animal” is used herein to mean humans and nonhuman animals ofboth sexes.

The terms “a”, “an” and “the” as used herein are defined to mean one ormore and include the plural unless the context is inappropriate.

“Proteins”, “peptides,” “polypeptides” and “oligopeptides” are chains ofamino acids (typically L-amino acids) whose alpha carbons are linkedthrough peptide bonds formed by a condensation reaction between thecarboxyl group of the alpha carbon of one amino acid and the amino groupof the alpha carbon of another amino acid. The terminal amino acid atone end of the chain (i.e., the amino terminal) has a free amino group,while the terminal amino acid at the other end of the chain (i.e., thecarboxy terminal) has a free carboxyl group. As such, the term “aminoterminus” (abbreviated N-terminus) refers to the free alpha-amino groupon the amino acid at the amino terminal of the protein, or to thealpha-amino group (imino group when participating in a peptide bond) ofan amino acid at any other location within the protein. Similarly, theterm “carboxy terminus” (abbreviated C-terminus) refers to the freecarboxyl group on the amino acid at the carboxy terminus of a protein,or to the carboxyl group of an amino acid at any other location withinthe protein.

Typically, the amino acids making up a protein are numbered in order,starting at the amino terminal and increasing in the direction towardthe carboxy terminal of the protein. Thus, when one amino acid is saidto “follow” another, that amino acid is positioned closer to the carboxyterminal of the protein than the preceding amino acid.

The term “residue” is used herein to refer to an amino acid (D or L) oran amino acid mimetic that is incorporated into a protein by an amidebond. As such, the amino acid may be a naturally occurring amino acidor, unless otherwise limited, may encompass known analogs of naturalamino acids that function in a manner similar to the naturally occurringamino acids (i.e., amino acid mimetics). Moreover, an amide bond mimeticincludes peptide backbone modifications well known to those skilled inthe art.

Furthermore, one of skill will recognize that, as mentioned above;individual substitutions, deletions or additions which alter, add ordelete a single amino acid or a small percentage of amino acids(typically less than 5%, more typically less than 1%) in an encodedsequence are conservatively modified variations where the alterationsresult in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. The following six groupseach contain amino acids that are conservative substitutions for oneanother:

-   -   1) Alanine (A), Serine (S), Threonine (T);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

When the peptides are relatively short in length (i.e., less than about50 amino acids), they are often synthesized using standard chemicalpeptide synthesis techniques. Solid phase synthesis in which theC-terminal amino acid of the sequence is attached to an insolublesupport followed by sequential addition of the remaining amino acids inthe sequence is a preferred method for the chemical synthesis of theantigenic epitopes described herein. Techniques for solid phasesynthesis are known to those skilled in the art.

Alternatively, the antigenic epitopes described herein are synthesizedusing recombinant nucleic acid methodology. Generally, this involvescreating a nucleic acid sequence that encodes the peptide or protein,placing the nucleic acid in an expression cassette under the control ofa particular promoter, expressing the peptide or protein in a host,isolating the expressed peptide or protein and, if required, renaturingthe peptide or protein. Techniques sufficient to guide one of skillthrough such procedures are found in the literature.

When several desired protein fragments or peptides are encoded in thenucleotide sequence incorporated into a vector, one of skill in the artwill appreciate that the protein fragments or peptides may be separatedby a spacer molecule such as, for example, a peptide, consisting of oneor more amino acids. Generally, the spacer will have no specificbiological activity other than to join the desired protein fragments orpeptides together, or to preserve some minimum distance or other spatialrelationship between them. However, the constituent amino acids of thespacer may be selected to influence some property of the molecule suchas the folding, net charge, or hydrophobicity. Nucleotide sequencesencoding for the production of residues which may be useful inpurification of the expressed recombinant protein may be built into thevector. Such sequences are known in the art. For example, a nucleotidesequence encoding for a poly histidine sequence may be added to a vectorto facilitate purification of the expressed recombinant protein on anickel column.

Once expressed, recombinant peptides, polypeptides and proteins can bepurified according to standard procedures known to one of ordinary skillin the art, including ammonium sulfate precipitation, affinity columns,column chromatography, gel electrophoresis and the like. Substantiallypure compositions of about 50 to 99% homogeneity are preferred, and 80to 95% or greater homogeneity are most preferred for use as therapeuticagents.

One of skill in the art will recognize that after chemical synthesis,biological expression or purification, the desired proteins, fragmentsthereof and peptides may possess a conformation substantially differentthan the native conformations of the proteins, fragments thereof andpeptides. In this case, it is often necessary to denature and reduceprotein and then to cause the protein to re-fold into the preferredconformation. Methods of reducing and denaturing proteins and inducingre-folding are well known to those of skill in the art.

The genetic constructs of the present invention include coding sequencesfor different proteins, fragments thereof, and peptides. The geneticconstructs also include epitopes or domains chosen to permitpurification or detection of the expressed protein, Such epitopes ordomains include DNA sequences encoding the glutathione binding domainfrom glutathione S-transferase, hexa-histidine, thioredoxin,hemagglutinin antigen, maltose binding protein, and others commonlyknown to one of skill in the art. The preferred genetic constructincludes the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 3 orfragments or conservative substitutions thereof. It is to be understoodthat additional or alternative nucleotide sequences may be included inthe genetic constructs in order to encode for the following: a) multiplecopies of the desired proteins, fragments thereof, or peptides; b)various combinations of the desired proteins, fragments thereof, orpeptides; and c) conservative modifications of the desired proteins,fragments thereof, or peptides, and combinations thereof. Still anotherpreferred protein of the present invention is human duox2 (SEQ ID NO: 2)protein, and fragments or conservative substitutions thereof, as encodedby SEQ ID NO: 1 and fragments or conservative substitutions thereof.Another preferred protein of the present invention is Ce Duox 1 (SEQ IDNO:4) protein and fragments or conservative substitutions thereof, asencoded by SEQ ID NO: 3 and fragments or conservative substitutionsthereof. The nucleotide sequences of the present invention may also beemployed to hybridize to nucleic acids such as DNA or RNA nucleotidesequences under high stringency conditions which permit detection, forexample, of alternately spliced messages.

The genetic construct is expressed in an expression system such as inNIH 3T3 cells using recombinant sequences in a pcDNA-3 vector(Invitrogen, Carlsbad, Calif.) to produce a recombinant protein.Preferred expression systems include but are not limited to Cos-7 cells,insect cells using recombinant baculovirus, and yeast. It is to beunderstood that other expression systems known to one of skill in theart may be used for expression of the genetic constructs of the presentinvention. A preferred protein of the present invention is referred toherein as human duox2, or fragments or conservative substitutionsthereof, which has the amino acid sequence set forth in SEQ ID NO:2, oran amino acid sequence having amino acid substitutions as defined in thedefinitions that do not significantly alter the function of therecombinant protein in an adverse manner. Another preferred protein ofthe present invention is Ce Duox 1 (SEQ ID NO: 4) or fragments orconservative substitutions thereof, as encoded by SEQ ID NO: 3 andfragments or conservative substitutions thereof.

Terminology

As described herein, the term “human duox2” refers to a proteincomprising an amino acid sequence as set forth in SEQ ID NO:2, or afragment or conservative substitution thereof, and encoded by thenucleotide sequence as set forth in SEQ ID NO:1, or a fragment orconservative substitution thereof Ce duox refers to duox from C. elegansor a fragment or conservative substitution thereof.

Construction of the Recombinant Gene

The desired gene is ligated into a transfer vector, such as pcDNA3, andthe recombinants are used to transform host cells such as Cos-7 cells.It is to be understood that different transfer vectors, host cells, andtransfection methods may be employed as commonly known to one ofordinary skill in the art. Two desired genes for use in transfection areshown in SEQ ID NO: 1, and SEQ ID NO: 3. For example,lipofectamine-mediated transfection and in vivo homologous recombinationis used to introduce the duox1 gene into NIH 3T3 cells.

The synthetic gene is cloned and the recombinant construct containingduox gene is produced and grown in confluent monolayer cultures of aCos-7 cell line. The expressed recombinant protein is then purified,preferably using affinity chromatography techniques, and its purity andspecificity determined by known methods.

A variety of expression systems may be employed for expression of therecombinant protein. Such expression methods include, but are notlimited to the following: bacterial expression systems, including thoseutilizing E. coli and Bacillus subtilis; virus systems; yeast expressionsystems; cultured insect and mammalian cells; and other expressionsystems known to one of ordinary skill in the art.

Transfection of Cells

It is to be understood that the vectors of the present invention may betransfected into any desired cell or cell line. Both in vivo and invitro transfection of cells are contemplated as part of the presentinvention. Preferred cells for transfection include but are not limitedto the following: fibroblasts (possibly to enhance wound healing andskin formation), granulocytes (possible benefit to increase function ina compromised immune system as seen in AIDS, and aplastic anemia),muscle cells, neuroblasts, stem cells, bone marrow cells, osteoblasts, Blymphocytes, and T lymphocytes.

Cells may be transfected with a variety of methods known to one ofordinary skill in the art and include but are not limited to thefollowing: electroporation, gene gun, calcium phosphate, lipofectamine,and fugene, as well as adenoviral transfection systems.

Host cells transfected with the nucleic acids represented in SEQ ID NO:1, and SEQ ID NO: 3, or fragments or conservative substitutions thereof,are used to express the proteins SEQ ID NO: 2 and SEQ ID NO: 4,respectively, or fragments or conservative substitutions thereof.

These expressed proteins are used to raise antibodies. These antibodiesmay be used for a variety of applications including but not limited toimmunotherapy against cancers expressing one of the duox proteins, foraffecting cuticle formation, and for detection, localization andmeasurement of the proteins shown in SEQ ID NO: 2 and SEQ ID NO: 4, orfragments or conservative substitutions thereof.

Purification and Characterization of the Expressed Protein

The proteins of the present invention can be expressed as a fusionprotein with a poly histidine component, such as a hexa histidine, andpurified by binding to a metal affinity column using nickel or cobaltaffinity matrices. The protein can also be expressed as a fusion proteinwith glutathione S-transferase and purified by affinity chromatographyusing a glutathione agarose matrix. The protein can also be purified byimmunoaffinity chromatography by expressing it as a fusion protein, forexample with hemagglutinin antigen. The expressed or naturally occurringprotein can also be purified by conventional chromatographic andpurification methods which include anion and cation exchangechromatography, gel exclusion chromatography, hydroxylapatitechromatography, dye binding chromatography, ammonium sulfateprecipitation, precipitation in organic solvents or other techniquescommonly known to one of skill in the art.

Methods of Assessing Activity of Expressed Proteins

Different methods are available for assessing the activity of theexpressed proteins of the present invention, including, but not limitedto, the proteins represented as SEQ ID NO: 2 and SEQ ID NO: 4,substituted analogs thereof, and fragments or conservative substitutionsthereof.

1. Assays of the Holoprotein and Fragments thereof for SuperoxideGeneration:

A. General Considerations.

These assays are useful in assessing efficacy of drugs designed tomodulate the activity of the enzymes of the present invention. Theholoprotein may be expressed in COS-7 cells, NIH 3T3 cells, insect cells(using baculoviral technology) or other cells using methods known to oneof skill in the art. Membrane fractions or purified protein are used forthe assay. The assay may require or be augmented by other cellularproteins such as p47phox, p67phox, and Rac1, as well as potentiallyother unidentified factors (e.g., kinases or other regulatory proteins).

B. Cytochrome C Reduction.

NADPH or NADH is used as the reducing substrate, in a concentration ofabout 100 μM. Reduction of cytochrome c is monitoredspectrophotometrically by the increase in absorbance at 550 nm, assumingan extinction coefficient of 21 mM⁻¹ cm⁻¹. The assay is performed in theabsence and presence of about 10 μg superoxide dismutase. Thesuperoxide-dependent reduction is defined as cytochrome c reduction inthe absence of superoxide dismutase minus that in the presence ofsuperoxide dismutase (Uhlinger et al. (1991) J. Biol. Chem. 266,20990–20997). Acetylated cytochrome c may also be used, since thereduction of acetylated cytochrome c is thought to be exclusively viasuperoxide.

C. Nitroblue Tetrazolium Reduction.

For nitroblue tetrazolium (NBT) reduction, the same general protocol isused, except that NBT is used in place of cytochrome c. In general,about 1 mL of filtered 0.25% nitrotetrazolium blue (Sigma, St. Louis,Mo.) is added in Hanks buffer without or with about 600 Units ofsuperoxide dismutase (Sigma) and samples are incubated at approximately37° C. The oxidized NBT is clear, while the reduced NBT is blue andinsoluble. The insoluble product is collected by centrifugation, and thepellet is re-suspended in about 1 mL of pyridine (Sigma) and heated forabout 10 minutes at 100° C. to solubilize the reduced NBT. Theconcentration of reduced NBT is determined by measuring the absorbanceat 510 nm, using an extinction coefficient of 11,000 M⁻¹ cm⁻¹. Untreatedwells are used to determine cell number.

D. Luminescence.

Superoxide generation may also be monitored with a chemiluminescencedetection system utilizing lucigenin (bis-N-methylpyridinium nitrate,Sigma, St. Louis, Mo.). The sample is mixed with about 100 μM NADPH(Sigma, St. Louis, Mo.) and 10 μM lucigenin (Sigma, St. Louis, Mo.) in avolume of about 150 μL Hanks solution. Luminescence is monitored in a96-well plate using a LumiCounter (Packard, Downers Grove, Ill.) for 0.5second per reading at approximately 1 minute intervals for a total ofabout 5 minutes; the highest stable value in each data set is used forcomparisons. As above, superoxide dismutase is added to some samples toprove that the luminescence arises from superoxide. A buffer blank issubtracted from each reading (Ushio-Fukai et al. (1996) J. Biol. Chem.271, 23317–23321).

E. Assays in Intact Cells.

Assays for superoxide generation may be performed using intact cells,for example, the duox-transfected NIH 3T3 cells. In principle, any ofthe above assays can be used to evaluate superoxide generation usingintact cells, for example, the duox-transfected NIH 3T3 cells. NBTreduction is a preferred assay method.

2. Assays of Truncated Proteins Comprised of Approximately theC-Terminal 265 Amino Acid Residues

While not wanting to be bound by the following statement, the truncatedprotein comprised of approximately the C-terminal 265 amino acidresidues is not expected to generate superoxide, and therefore,superoxide dismutase is not added in assays of the truncated protein.Basically, a similar assay is established and the superoxide-independentreduction of NBT, cytochrome c, dichlorophenolindophenol, ferricyanide,or another redox-active dye is examined.

Nucleotides and Nucleic Acid Probes

The nucleotide sequences SEQ ID NO: 1 and SEQ ID NO: 3, as well asfragments or conservative substitutions thereof, and PCR primerstherefor, may be used, respectively, for localization, detection andmeasurement of nucleic acids related to SEQ ID NO: 1 and SEQ ID NO: 3,as well as fragments or conservative substitutions thereof SEQ ID NO 1is also known as a nucleotide sequence encoding human duox2 in thisapplication. SEQ ID NO: 3 is also known as a nucleotide sequenceencoding Ce duox 1 in this application.

The nucleotide sequences SEQ ID NO: 1, SEQ ID NO: 3, as well asfragments or conservative substitutions thereof, may be used to createprobes to isolate larger nucleotide sequences containing the nucleotidesequences SEQ ID NO: 1, SEQ ID NO: 3, respectively. The nucleotidesequences SEQ ID NO: 1, SEQ ID NO: 3, as well as fragments orconservative substitutions thereof, may also be used to create probes toidentify and isolate duox proteins in other species.

The nucleic acids described herein include messenger RNA coding forproduction of SEQ ID NO: 2, SEQ ID NO: 4, and fragments thereof. Suchnucleic acids include but are not limited to cDNA probes. These probesmay be labeled in a variety of ways known to one of ordinary skill inthe art. Such methods include but are not limited to isotopic andnon-isotopic labeling. These probes may be used for in situhybridization for localization of nucleic acids such as mRNA encodingfor SEQ ID NO: 2, and SEQ ID NO: 4, and fragments or conservativesubstitutions thereof. Localization may be performed using is situhybridization at both ultrastructural and light microscopic levels ofresolution using techniques known to one of ordinary skill in the art.

These probes may also be employed to detect and quantitate nucleic acidsand mRNA levels using techniques known to one of ordinary skill in theart including but not limited to solution hybridization.

Antibody Production

The proteins shown in SEQ ID NO: 2, SEQ ID NO: 4 SEQ ID NO: 31 and SEQID NO: 32, or fragments or conservative substitutions thereof, arecombined with a pharmaceutically acceptable carrier or vehicle toproduce a pharmaceutical composition and administered to animals for theproduction of polyclonal antibodies using methods known to one ofordinary skill in the art. The preferred animals for antibody productionare rabbits and mice. Other animals may be employed for immunizationwith these proteins or fragments thereof. Such animals include, but arenot limited to the following; sheep, horses, pigs, donkeys, cows,monkeys and rodents such as guinea pigs and rats.

The terms “pharmaceutically acceptable carrier or pharmaceuticallyacceptable vehicle” are used herein to mean any liquid including but notlimited to water or saline, oil, gel, salve, solvent, diluent, fluidointment base, liposome, micelle, giant micelle, and the like, which issuitable for use in contact with living animal or human tissue withoutcausing adverse physiological responses, and which does not interactwith the other components of the composition in a deleterious manner.

The pharmaceutical compositions may conveniently be presented in unitdosage form and may be prepared by conventional pharmaceuticaltechniques. Such techniques include the step of bringing intoassociation the active ingredient and the pharmaceutical carrier(s) orexcipient(s). In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example, sealed ampules and vials, and may be stored ina freeze-dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, for example, water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletscommonly used by one of ordinary skill in the art.

Preferred unit dosage formulations are those containing a dose or unit,or an appropriate fraction thereof, of the administered ingredient. Itshould be understood that in addition to the ingredients, particularlymentioned above, the formulations of the present invention may includeother agents commonly used by one of ordinary skill in the art.

The pharmaceutical composition may be administered through differentroutes, such as oral, including buccal and sublingual, rectal,parenteral, aerosol, nasal, intramuscular, subcutaneous, intradermal,and topical. The pharmaceutical composition of the present invention maybe administered in different forms, including but not limited tosolutions, emulsions and suspensions, microspheres, particles,microparticles, nanoparticles, and liposomes. It is expected that fromabout 1 to 7 dosages may be required per immunization regimen. Initialinjections may range from about 0.1 μg to 1 mg, with a preferred rangeof about 1 μg to 800 μg, and a more preferred range of fromapproximately 25 μg to 500 μg. Booster injections may range from 0.1 μgto 1 mg, with a preferred range of approximately 1 μg to 800 μg, and amore preferred range of about 10 μg to 500 μg.

The volume of administration will vary depending on the route ofadministration and the size of the recipient. For example, intramuscularinjections may range from about 0.1 ml to 1.0 ml.

The pharmaceutical composition may be stored at temperatures of fromabout 4° C. to −100° C. The pharmaceutical composition may also bestored in a lyophilized state at different temperatures including roomtemperature. The pharmaceutical composition may be sterilized throughconventional means known to one of ordinary skill in the art. Such meansinclude, but are not limited to filtration, radiation and heat. Thepharmaceutical composition of the present invention may also be combinedwith bacteriostatic agents, such as thimerosal, to inhibit bacterialgrowth.

Adjuvants

A variety of adjuvants known to one of ordinary skill in the art may beadministered in conjunction with the protein in the pharmaceuticalcomposition. Such adjuvants include, but are not limited to thefollowing: polymers, co-polymers such aspolyoxyethylene-polyoxypropylene copolymers, including blockco-polymers; polymer P1005; Freund's complete adjuvant (for animals);Freund's incomplete adjuvant; sorbitan monooleate; squalene; CRL-8300adjuvant; alum; QS 21, muramyl dipeptide; trehalose; bacterial extracts,including mycobacterial extracts; detoxified endotoxins: membranelipids; or combinations thereof.

Monoclonal antibodies can be produced using hybridoma technology inaccordance with methods well known to those skilled in the art. Theantibodies are useful as research or diagnostic reagents or can be usedfor passive immunization. The composition may optionally contain anadjuvant.

The polyclonal and monoclonal antibodies useful as research ordiagnostic reagents may be employed for detection and measurement of SEQID NO: 2, SEQ ID NO: 4, SEQ ID NO: 31 and SEQ ID NO: 32, and fragmentsor conservative substitutions thereof. Such antibodies may be used todetect these proteins in a biological sample, including but not limitedto samples such as cells, cellular extracts, tissues, tissue extracts,biopsies, tumors, and biological fluids. Such detection capability isuseful for detection of disease related to these proteins to facilitatediagnosis and prognosis and to suggest possible treatment alternatives.

Detection may be achieved through the use of immunocytochemistry, ELISA,radioimmunoassay or other assays as commonly known to one of ordinaryskill in the art. The duox proteins, including the hduox2 and Ce-duoxproteins of the present invention, or fragments or conservativesubstitutions thereof, may be labeled through commonly known approaches,including but not limited to the following: radiolabeling, dyes,magnetic particles, biotin-avidin, fluorescent molecules,chemiluminescent molecules and systems, ferritin, colloidal gold, andother methods known to one of skill in the art of labeling proteins.

Administration of Antibodies

The antibodies directed to the proteins shown as SEQ ID NO: 2, SEQ IDNO: 4, SEQ ID NO: 31, SEQ ID NO: 32, or directed to fragments orconservative substitutions thereof, may also be administered directly tohumans and animals in a passive immunization paradigm. Antibodiesdirected to extracellular portions of SEQ ID NO: 2, and SEQ ID NO: 4,bind to these extracellular epitopes. Attachment of labels to theseantibodies facilitates localization and visualization of sites ofbinding. Attachment of molecules such as ricin or other cytotoxins tothese antibodies helps to selectively damage or kill cells expressingSEQ ID NO: 2, and SEQ ID NO: 4, or fragments thereof.

Kits

The present invention includes kits useful with the antibodies, nucleicacids, nucleic acid probes, labeled antibodies, labeled proteins orfragments thereof for detection, localization and measurement of SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or combinations andfragments or conservative substitutions thereof.

Kits may be used for immunocytochemistry, in situ hybridization,solution hybridization, radioimmunoassay, ELISA, Western blots,quantitative PCR, and other assays for the detection, localization andmeasurement of these nucleic acids, proteins or fragments thereof usingtechniques known to one of skill in the art.

The nucleotide sequences shown in SEQ ID NO: 1, and SEQ ID NO: 3, orfragments thereof, may also be used under high stringency conditions todetect alternately spliced messages related to SEQ ID NO: 1, and SEQ IDNO: 3, or fragments thereof, respectively.

The diagnostic kits may measure or detect the relative expression of theduox proteins described herein (i.e. human duox1 and/or human duox2 andce-duox).

Fragments of SEQ ID NO: 1, and SEQ ID NO: 3, containing the relevanthybridizing sequence can be synthesized onto the surface of a chiparray. RNA samples, e.g., from tumors, are then fluorescently tagged andhybridized onto the chip for detection. This approach may be useddiagnostically to characterize tumor types and to tailor treatmentsand/or provide prognostic information. Such prognostic information mayhave predictive value concerning disease progression and life span, andmay also affect choice of therapy.

The present invention is further illustrated by the following examples,which are not to be construed in any way as imposing limitations uponthe scope thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof, which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention.

EXAMPLE 1

Cloning of cDNA for Human Duox 2

A 535-base portion of an expressed sequence tag (EST zc92h03.r1; Genbankaccession no. W52750) from human pancreatic islet was identified usingthe amino-acid sequence of human gp91phox as a query in a Blast search.The bacterial strain #595758 containing the EST sequence zc92h03.r1 inthe pBluescript SK-vector was purchased from ATCC (Rockville, Md.). TheDNA was sequenced using primers to T7 and T3 vector promoters as well assequence-specific internal primers. The EST encoded a 440 amino acidpartial cDNA exhibiting 24.4% identity to gp91phox, including a stopcodon corresponding to the C-terminus of gp91phox. 5′- and 3′-RACE werecarried out using human adult pancreas mRNA (Clontech, Palo Alto,Calif.) with the 5′ RACE kit for Rapid Amplification of cDNA Endsversion 2.0 (Gibco BRL, Gaithersburg, Md.). PCR was done with specificprimers: 5′-RACE: Primer 1, 5′-GAAGTGGTGGGAGGCGAAGACATA-3′ (SEQ IDNO:5); Primer 2, 5′-CCTGTCATACCTGGGACGGTCTGG-3′ (SEQ ID NO:6); Primer 3,5′-GAGCACAGTGAGATGCCTGTTCAG-3′ (SEQ ID NO:7); Primer 4,5′-GGAAGGCAGCAGAGAGCAATGATG-3′ (SEQ ID NO:8); Primer 5,5′-AGGTGGGATGCGGATGTTGAG-3′ (SEQ ID NO:9) (for nested PCR); 3′-RACEPrimer 6, 5′-ACATCTGCGAGCGGCACTTCCAGA-3′ (SEQ ID NO:10); Primer 7,5′-AGCTCGTCAACAGGCAGGACCGAGC-3′ (SEQ ID NO:11); Primer 8,5′-TCTCCATCAGAATCCACCTTAGGC-3′ (SEQ ID NO:12) (for nested PCR). Tocomplete the sequence, 5′-RACE was carried out using human thyroidMarathon-ready cDNA (Clontech, Palo Alto, Calif.) with primer 3 andadapter primer AP1, and primer 5 and adapter primer AP2. Theseprocedures resulted in an additional 3.7 kb 5′ region and a 1.5 kb 3′region.

The cDNA for h-Duox2 showed a 4647 base pair open reading frame(Genebank #AF267981) that is predicted to encode a protein of 1548 aminoacids (175 kDa), and contained a consensus Kozak sequence, GGCATGC (SEQID NO: 13), at the translation start codon. The Duox2 cDNA sequence is alarger form of a gp91phox homolog previously identified as anNADPH-oxidase in thyroid and termed p138^(Tox); the latter sequence didnot contain the a peroxidase homology domain (Dupuy et al., 1999).h-Duox1 and h-Duox2 were 77% identical at the amino acid level.

EXAMPLE 2

Identification of Genes for Ce-Duox1 and Ce-Duox2, Cloning of the cDNAfor Ce-Duox1

A BLAST search using the cDNA sequence of human gp91phox identified twoputative homologues (Genbank #s AF043697 and AF003130) in the genomicsequence of C. elegans, both near the end of chromosome I and separatedby ˜6 Kb. Based on the gene sequence, PCR primers were designed toamplify two overlapping portions of the Ce-Duox1 gene, one extendingfrom the 5′ end and one extending from the 3′ end. Primers were5′-ATTCGTCGACAAATGCGCTCAAAACATGTGCTGT-3′ (SEQ ID NO: 14) and5′-AACTTTGTGGATCAAAGTTAGCG-3′ (SEQ ID NO: 15) for the 5′ region, and5′-TTGGATTAGCATTTTGCTATGGAA-3′ and (SEQ ID NO: 16)5′-GAGCGGCCGCGAACGTTTCAAAGCGATGTGCA-3′ (SEQ ID NO: 17) for the 3′region. PCR was carried out using a random primed C. elegans cDNAlibrary in 1ACT (obtained from R. Barstead, Oklahoma Medical ResearchFoundation) under the following conditions: denaturation at 95° C. for30 seconds; annealing at 59° C. for 30 seconds; extension at 72° C. for1 minute. The 5′ piece and the 3′ piece were digested with Dra III andligated to produce the full length Ce-Duox1 cDNA. The full lengthCe-Duox1 cDNA was inserted into the pBluescript SK-vector and wassequenced using T7 and T3 vector primers and sequence specific primers.

Duox Homologs in C. elegans A BLAST search of the C. elegans genomicdatabase using as a query the protein sequence of gp91phox identifiedtwo homologous genes contained in cosmids F56C11 and F53G12. TheCe-Duox1 conceptual transcript (Genebank #AF043697) is predicted to be8197 bp before splicing, to contain 19 exons, and to encode a protein of1506 amino acids. Cloning of the cDNA for Ce-Duox1 (Genebank #AF229855)revealed a cDNA of 4491 bp (1497 amino acids), which differed somewhatfrom the conceptual cDNA obtained from the gene structure due toinaccuracies in the predicted intron-exon junctions. The secondtranscript, Ce-Duox2 (Genebank #AF043697), is predicted to be 5308 bpbefore splicing, to contain 16 exons, and to encode a 1313 amino acidprotein. Alignment by homology of the genomic sequences of Ce-Duox1 andCe-Duox2 identified two new exons 5′ of the first predicted exon ofDuox2 that were highly homologous to the second and third exons ofDuox1, but an exon of Duox2 homologous to exon1 of Duox1 could not beidentified by homology. The predicted amino acid sequences of bothCe-Duox1 and Ce-Duox2 show approximately 30% identity with h-Duox1 andh-Duox2 (FIGS. 1 and 2A). Ce-Duox1 also contains the same domains ash-Duox1/2 (see below) and is roughly the same size. However, Ce-Duox2contains a stop codon which should eliminate the extreme C-terminalportion of the protein, which includes a segment of the pyridinenucleotide binding site. Thus, while Ce-Duox2 should contain intactperoxidase and calmodulin-like domains, it is not predicted to encode afunctioning NADPH-oxidase domain (see FIG. 1). Except for thisC-terminal region, Ce-Duox2 is 94% identical to Ce-Duox1 at the aminoacid level. Both Ce-Duox1 and Ce-Duox2 are located near the end ofchromosome I, separated by only 6 kb and in opposite orientations. Thehigh degree of sequence identity and retention of intron structure (datanot shown), as well as the location of both near the end of a chromosomeare consistent with a recent gene duplication.

EXAMPLE 3

Analysis of Primary Structure; Domain Organization and SequenceComparisons among gp91phox, h-Duox2, Ce-Duox1 and Ce-Duox2.

Export signal sequences were predicted according to Nielsen et al.,1997. Transmembrane alpha helices were predicted according to Sonnhammeret al., 1998. Both methods are available on the internet at the Centerfor Biological Sequence Analysis (http://www.cbs.dtu.dk/services/).Multiple sequence alignments phylogenetic analysis were carried outusing the clustal method, using Megalign software (DNASTAR).

The domain structure and transmembrane regions in gp91phox, h-Duox1,hDuox2, Ce-Duox1 and Ce-Duox2 are diagrammed in FIG. 1. Duox enzymes arehomologous to gp91phox in their C-termini (seehttp://www.biochem.emory.edu/Lambeth/gp91_homology.pdf for an alignmentof these regions). Nox1 (Suh et al., 1999), which is the same size asgp91phox, is more closely related to gp91phox (54% identical) than isthe NADPH-oxidase domain of hDuox1 or hDuox2 (˜26 % identical togp91phox). However, h-Duox1 and hDuox2 are more closely related toCe-Duox1 within the NADPH-oxidase domain (˜39% identical). Within theputative FAD binding regions and NADPH binding regions, homologs shareconsiderably higher homology, ranging from 60% to 90%, depending on theregion. This includes the canonical dinucleotide binding helix GXGXXP(SEQ ID NO: 18). In gp91phox, Nox1, h-Duox1, and h-Duox2 this sequenceis followed by F, which is present in many NADPH-specific flavoproteins,while in the C. elegans proteins, F is conservatively replaced with Y.

Duox proteins have additional regions that are not present in gp91phox.A central region contains two EF hand calcium binding sequences, asindicated in FIG. 1. The canonical residues involved in calcium ligationare well conserved in h-Duox1 and h-Duox2, but are poorly conserved inCe-Duox1 and Ce-Duox2, suggesting that the function of this region mayhave evolved away from calcium binding in nematodes.

Surprisingly, the N-terminal third of Duox proteins is homologous toperoxidases including myeloperoxidase, eosinophil peroxidase, thyroidperoxidase, lactoperoxidase and sea urchin ovoperoxidases (FIGS. 2A and2B). Overall, the identity with peroxidases within the entire region isapproximately 20%, but sub-regions show considerably higher homology.The Duox enzymes represent a distinct group within the peroxidase family(FIG. 2B), and phylogenetically, this group is marginally more closelyrelated to sea urchin ovoperoxidases. Within the peroxidase homologyregion, only 2 of the 12 cysteine residues involved in the sixintra-chain disulfide bonds, which are conserved in the four homologousmammalian peroxidases, are present in Duox proteins (not shown). Inaddition, the asparagine-linked glycosylation sites found in MPO are notpresent in Ce-Duox1 or Ce-Duox2. A calcium binding site in MPO(aspartate 263 and residues 335 to 341, superior double bar in FIG. 2A)(Zeng and Fenna, 1992) is well conserved in the Duox family proteins,including 3 of the 4 candidate calcium liganding residues (filledtriangles).

The extreme N-terminal 21 amino acids of Ce-Duox1 contains a secretorysignal peptide sequence (FIG. 1), implying that the N-terminalperoxidase domain is in a compartment that is transmembrane to thecytosol (e.g., extracellular or within a secretory vesicle). Inaddition, hydropathy plots reveal that the proteins contain a highlyhydrophobic region corresponding to the N-terminal third of gp91phox.This region can be modelled as a cluster of 6 transmembrane alphahelices, as indicated in FIG. 1. An additional transmembrane helicalregion is present between the peroxidase homology domain and thecalmodulin-like domain.

EXAMPLE 4

PCR Detection of mRNA for Human Duox, Tissue Distribution of h-DuoxmRNA.

Based on the cloned h-Duox1 and hDuox2 cDNA sequence, we designedspecific primers (Duox1: 5′-GCAGGACATCAACCCTGCACTCTC-3′ (SEQ ID NO:19);5′-CTGCCATCTACCACACGGATCTGC-3′ (SEQ ID NO:20); Duox2:5′-GCCCTCAACCTAAGCAGCTCACAACTG-3′ (SEQ ID NO:21);5′-GAGCACAGTGAGATGCCTGTTCAG-3′) (SEQ ID NO:22) which were used todetermine the tissue expression patterns of Duox1 and Duox2 using HumanMultiple Tissue PCR Panels and human thyroid gland Marathon-Ready cDNA(Clontech, Palo Alto, Calif.). PCR conditions were: 95° C. for 30 s, 65°C. for 20 s, 72° C. for 30 s, 35 cycles.

h-Duox1 mRNA was distributed among a variety of adult tissues, withhighest expression in lung and thyroid, but with significant expressionalso seen in placenta, testis, and prostate with detectable expressionin pancreas and heart. h-Duox1 mRNA was also widely expressed in fetaltissues, where it was abundant in lung. In addition, we observedsignificant expression in a variety of fetal tissues and in adult colon,with detectable expression in kidney, liver, lung, pancreas, prostateand testis.

h-Duox2 mRNA was distributed among a variety of adult tissues, withhighest expression in colon, testis, pancreas and thyroid. h-Duox2mRNAwas also widely expressed in fetal tissues, where it was abundant inlung, liver, kidney, and heart, and thyroid. We also observedsignificant expression in fetal skeletal muscle and thymus.

EXAMPLE5

RNA Interference (RNAi) in C. elegans Phenotypes of C. elegans RNAiCe-Duox Animals

To gain insights regarding the biological function of Duox enzymes, weused the reverse genetic tool, RNA interference (RNAi), to “knock out”Duox in C. elegans (Fire et al., 1998). This technique involvesinjection of double stranded RNA (dsRNA) encoding a segment of Ce-Duox1or Ce-Duox2 into gonads of C. elegans wild type hermaphrodites. Injectedanimals were then allowed to lay eggs, the harvested eggs were allowedto develop, and the progeny were observed for phenotypes. This procedurespecifically diminishes or eliminates the expression of the gene ofinterest.

RNA was transcribed from either pBluescript.Duox2, pBluescript.E17Duox1or pBluescript.E 18+19Duox1. For pBluescript.Duox2, Exon 10 of Ce-Duox2was amplified by PCR from genomic DNA using the forward primer5′-GCTAGAGCTCTTCAGTTTGCTATGGAATTGGC-3′ (SEQ ID NO:23) and reverse primer5′-CATAAAGGATGAGGAGAATTCTGTG-3′ (SEQ ID NO:24). The 457-bp fragmentgenerated was digested with SstI and EcoRI and subcloned intopBluescript. For pBluescript.E17Duox1, Exon 17 of Duox1 was amplified byPCR from genomic DNA using the forward primer5′-GCTAGAGCTCGGCTACTACTACGTTGTTGGACC-3′ (SEQ ID NO:25) and the reverseprimer 5′-GACTGAAGGACTTGTGGAACGTCTGAGTGAC-3′ (SEQ ID NO:26). The 659 bpfragment generated was digested with SstI and EcoRI and sub cloned intopBluescript. For pBluescript.E18+19Duox1, Exons 18 and 19 of Ce-Duox1were amplified by PCR from a randomly primed C. elegans cDNA library(obtained from R. Barstead, Oklahoma Medical Research Foundation) usingthe forward primer 5′-GCTAGAGCTCACATTTGCGAGAAGCACTTCCG-3′ (SEQ ID NO:27) and the reverse primer 5′-GTGTGAATTCAGCGATGTGCAAATGAAGGAGC-3′ (SEQID NO: 28). The 266 bp fragment generated was digested with SstI andEcoRI and subcloned into pBluescript. Plasmids were linearized witheither Sst1 or EcoR1 and transcription was carried out using T3 and T7RNA polymerase (Promega) in separate reactions. Sense and antisensesingle-stranded RNAs were combined in equal concentrations, andincubated for 10 min at 68° C. followed by a 30 min incubation at 37° C.to form double stranded RNA (dsRNA). dsRNAs were injected into thegonads of N2 hermaphrodite C. elegans as described in detail in thefollowing paragraph. Injected animals were allowed to recover and layeggs for ˜20 h after injection, transferred to individual plates, andallowed to lay eggs for a second 24 h period. The F1 progeny resultingfrom this second period of egg laying were evaluated. Phenotypes wereobserved in >90% of F1 animals.

Phenotypes of C. elegans RNAi Ce-Duox Animal

dsRNA corresponding to three distinct regions of Ce-Duox1 and Duox2 wereused in separate experiments. The first two correspond to regions ofidentity between Ce-Duox1 and Ce-Duox2 and are predicted to block theexpression of both forms of Duox. The third dsRNA corresponds to theextreme C-terminus of Ce-Duox1, which does not have a counterpart inCe-Duox2, and therefore blocks only the expression of Ce-Duox1. Allthree dsRNA forms resulted in the same range of phenotypes. In replicateexperiments, the percentage of animals exhibiting any given phenotypewas somewhat variable, probably due to differences in amount of RNAi orsite of injection. However, in a typical experiment, greater than 90% ofthe animals were affected by one or more phenotypes. In a typicalexperiment, phenotypes included the presence of large superficialblisters (˜50% of animals) and short or “dumpy” animals (˜35% ofanimals), and animals with retained eggs or larvae (not shown). Inaddition, while wild type animals showed a dark appearance, more than80% of RNAi animals were translucent. Around half of RNAi animals showedan inability to move on plates in a normal serpentine manner: affectedanimals were either completely paralyzed or moved only the anteriorregion, clearing a localized swath of E. coli in the vicinity of thehead.

Similar phenotypes in C. elegans have been described previously and areassociated with mutations in the collagen biosynthetic pathway (Levy etal., 1993; Kramer, 1997; Johnstone, 2000). Several genes that encodecuticle collagens, when mutated, result in Bli (“blister”), Dpy(“dumpy”, short fat worm), Rol (“roller”, helical motion instead of aflat, sinusoidal motion), or Sqt (“squat”, generally rollers as larvaeand dumpy as adults) phenotypes. The genetics of this process arecomplex, since for some genes, different mutations in the same gene giverise to different phenotypes, and sometimes the phenotypes are combined(e.g. “dumpy roller”) in nematodes, collagen along with several otherproteins provide the major components of cuticle, an extracellularmatrix which acts as an exoskeleton.

In a global analysis of expression of all C. elegans genes usingoligonucleotide arrays (Hill et al., 2000), Ce-Duox1 was expressed atlow levels (consistent with its exclusive expression in hypodermalcells) in a stage-specific manner. Expression occurred in a cyclicpattern peaking during the embryonic stage and at 36 hours,corresponding to the peak expression of other genes (Johnstone, I. L.,2000) related to collagen/cuticle biosynthesis (col-14, dpy-2, -7, -10,and sqt-3). A second set of collagen/cuticle-related genes (bli-1, -2,col-2, -6, -17, -35, -36, -37, -41, dyp-13, sqt-1, and rol-6, -8) alsoshow peak expression at 36 hours. No significant expression of Ce-Duox2was seen at any stage. Thus, these data are consistent with a functionof Ce-Duox1 in cuticle biogenesis.

EXAMPLE 6

Generations of Transgenic Nematodes to Study Ce-Duox1 Expression

DNA from pPD96.62PRODuox1B was mixed with myo-3-GFP DNA (kindly providedby A. Fire, Carnegie Institute of Embryology, Baltimore, Md.) andinjected into wild type or rol-6(su1006) young adult hermaphrodites(Mello and Fire, 1995). Transformants were identified by screening theF1 progeny under a fluorescence dissecting microscope for green bodywall muscle. These green glowing animals were stained forβ-galactosidase expression, as described below. Although pPD96.62 wasexpected to have driven both n-galactosidase and green fluorescentprotein (GFP) expression, no fluorescence was observed outside of bodywall muscle (the site of expression of the marker Myo-3-GFP).pPD96.62PRODuox1B was prepared as follows: a 3389 bp fragment wasamplified by long range PCR from C. elegans genomic DNA using theforward primer 5′-AGTCGAAGCTTAGCATGTCAAAGTCCGGAGTTCAGT-3′ (SEQ ID NO:29)and the reverse primer 5′-CTAGTGGATCCGCATTGCTCGTGCGCCTTAGAGTTT-3′ (SEQID NO:30). The fragment included the start methionine of Ce-Duox1 and 5′untranslated sequence. The fragment was digested with HindIII and BamHIand then subcloned into pPD96.62. This construct results in the Ce-Duox1promoter region (3389 bp) and the start methionine being inserted 5′ ofE. coli lacZ gene fused to the green fluorescence protein (GFP) reportedgene.

β-Galactosidase Staining

Staining was used to detect expression of the gfp:lacZ fusion protein intransgenic worms carrying pPD96.62PRODuox1B. Reagent preparation andfixation were performed as described by Fire (1993). Vectors alsoincorporated a nuclear localization peptide at the N-terminus ofβ-galactosidase. This allows predominant staining in the nuclei ofexpressing cells and facilitates their identification. Nematodes wereplaced into individual wells of an eight well microscope slide with ˜15μl of distilled water and dried under vacuum for 2–3 min. Acetone wascontinuously dripped onto the dried animals for 2 min. The slide wasplaced in an uncovered humidity chamber, but kept dry. 10 μl ofβ-galactosidase stain (Fire, 1993) was layered onto each well as soon asthe acetone had completely evaporated and the lid to the humiditychamber was replaced. The nematodes were then incubated at roomtemperature for several hours, washed several items in phosphatebuffered saline, and then observed with a compound microscope.

Cellular Expression of Ce-Duox1

The cellular location of Ce-Duox1 in C. elegans was determined by doublestaining with antibodies to Ce-Duox1 and to myosin A (a marker for bodywall muscle cells). Ce-Duox1 was seen in larval animals in thehypodermal layer of cells immediately overlying the myosin A-containingmuscle cells, and was only faintly detectable in hypodermal cells thatdid not overlie muscle quadrants. In adult animals, Ce-Duox1 was poorlydetected (not shown). The strong signal seen in larval animals waseliminated using anti-Ce-Duox1 antibody that had been preincubated withCe-Duox1 peptide.

EXAMPLE 7

Antibody Production and Purification

A 16 amino acid peptide corresponding to residues 340–355 of Ce-Duox1was synthesized by the Emory Microchemical Facility and coupled usinggluteraldehyde to keyhole limpet hemocyanin (KLH). Rabbit antibody wasprepared against KLH-conjugated peptide by Lampire BiologicalLaboratories (Pipersville, Pa.) using standard protocols. Peptide (30mg) was coupled to 1 ml of Affi-Gel 10 (Bio-Rad) for antibodyimmunopurification; 2 ml of serum was dialyzed against PBS and wasloaded onto the Affi-Gel column preequilibrated with PBS. The column waswashed with 10 ml PBS containing 1M NaCl. 0.5 ml factions of antibodywere eluted with 0.1M glycine-HCl (pH 2.4) and were immediatelyneutralized with TRIS, pH 9. Fractions containing the highestconcentration of protein were used in immunofluorescence experiments.

EXAMPLE 8

Western Blot

Nematodes were washed with M9 buffer, suspended in 0.5 ml sonicationbuffer (10 mM Tris HCl, pH 7.4, 1 mM EDTA, 1 mM phenylmethanesulfonylfluoride), and sonicated 4×20 s. Protein was determined with theBradford assay using bovine serum albumin as a standard. 10 μg of wholeanimal extract was loaded onto a 10% SDS-page gel which was thentransferred to Immobilon-P membrane (Millipore). The blot was blockedfor 1 hour in a solution of 5% nonfat powdered milk and 0.05% Tween inPBS. The antibody to Ce-Duox1 was added in a 1 to 2000 dilution,incubated overnight, and the membrane was washed 3 times for 15 min withblocking solution. The blot was then developed using the SuperSignalChemiluminescent Kit from Pierce (Rockford, Ill.). A western blot of C.elegans protein extract showed a single band with a molecular weight of˜180,000 (data not shown).

EXAMPLE 9

Indirect Inmmunofluorescence

Inmmunofluorescence staining of C. elegans was carried out as in Benianet al., 1996. Mouse antibody to Myosin A was a gift from D. Miller(Miller et al., 1983). Goat anti-rabbit rhodamine-conjugated antibodyand goat anti-mouse FITC-conjugated antibody were used as secondaryantibodies for the detection of Ce-Duox1 and Myosin A respectively. Todetermine non-specific binding of the Ce-Duox1 antibody, a 10 fold molarexcess of Ce-Duox1 peptide was added to neutralize the antibody.Microscopy was carried out using Zeiss 510 laser scanning confocalmicroscope.

EXAMPLE 10

Preparation of Dityrosine Standard

Dityrosine standard was synthesized and purified as in Abdelrahim etal., 1997 with minor modifications. Reaction products were dissolved inacidified methanol, were filtered, and directly applied to the CP-11cellulose phosphate, eliminating the rotary evaporation step. Sampleswith absorption properties characteristic of dityrosine were pooled andfreeze dried. For mass spectrometry, the 1 ml of dityrosine standard(0.77 mg/ml) was added to 1 ml of methanol:water (1:1) in 0.1% aceticacid.

Analysis of Dityrosine and Trityrosine

Nematodes were washed with M9 buffer, suspended in 0.5 ml sonicationbuffer (10 mM Tris HCl, pH 7.4, 1 mM EDTA, 1 mM phenylmethanesulfonylfluoride), and sonicated 4×20 s. Protein was determined with theBradford assay using bovine serum albumin as a standard. Whole wormextracts were lyophilized and resuspended in 6 N HCl. Samples werehydrolyzed for 24 h at 110° C. under vacuum, dried under vacuum andresuspended in the mobile phase for analysis by high performance liquidchromatography (HPLC) on a C18 column (0.46×26 cm, Fisher) using aDionex AGP-1 HPLC instrument. The mobile phase consisted of 0.1 M KH₂PO₄adjusted to pH 3.8 with 0.1 M phosphoric acid at a flow rate of 1ml/min. The column eluent was monitored by fluorescence with anexcitation 305–395 nm bandpass filter and an emission filter at 450 nmwith a bandpass of 40 nm. To verify the identity of dityrosine,authentic dityrosine standard was added to some samples and an increasein the intensity of the putative dityrosine band was observed (data notshown).

Spectroscopic Properties of Di- and Trityrosine

HPLC purified samples of dityrosine and trityrosine from both C. elegansextracts and peroxidase domain cross-linking reactions were lyophilizedand resolubilized in either 0.1 M HCl (3 ml) or 0.1 M NaOH (3 ml).Fluorescence excitation and emission spectra were obtained with aPerkin-Elmer LS-5B Luminescence Spectrometer.

Mass Spectrometry

Mass spectrometry was preformed on a PE sciex API 3000 triple quadrupolemass spectrometer equipped with a turboionspray source. Dried dityrosinestandard (20 mg) was reconstituted in 200 μl of H₂O. A 50 μl aliquot ofthis was diluted to a final volume of 1 ml with 950 μl of 5 mM ammoniumacetate in MeOH and 1% acetic acid. This solution infused at a flow rateof 5 μl min⁻¹. The ionspray needle was held at +550V and −4500V forpositive and negative ion analysis, respectively. These experimentsidentified the singly protonated (positive ion mode) and deprotonated(negative ion mode) species of the standard to be m/z 361.3 and 359.3respectively as predicted.

Standard and total protein acid hydrolysate from C. elegans wereanalyzed by reverse phase LC-MS/MS. A 50 μl volume of sample wasinjected onto a 15 cm×2.1 mm Supelco Discovery C18 column at a flow rateof 300 μl min⁻¹. Solvent A was 99:1 H₂O/acetic acid and solvent B was99:1 MeOH/acetic acid both containing 5 mM ammonium acetate. The columnwas directly infused into the ion source of the mass spectrometeroperating in positive ion mode. The column was pre-equilibrated with100% A for 6 min followed by sample injection. The column was thenwashed with 100% A for 4 min and eluted with a 1 min linear gradient to100% B, followed by a 4 min wash with 100% B. For these experiments boththe precursor ions (as above for dityrosine; m/z 540.4/538.4 fortrityrosine) and structurally distinctive breakdown ions were monitored.The transitions monitored for dityrosine were the neutral loss of bothcarboxyl groups, the neutral loss of both carboxyl groups and one aminogroup, and the neutral loss of both carboxyl groups and both aminogroups (m/z 269.4, 252.2, and 235.0 respectively). For trityrosine, thetransitions monitored were the neutral loss of a carboxyl groups, theneutral loss of a carboxyl group and one amino group, the neutral lossof two C-termini, and the neutral loss of two carboxyl groups and twoamino groups (m/z 494.3, 477.2, 448.2, and 431.2 respectively).

Absence of Tyrosine Cross-linking in RNAi Nematodes

Cross-linking of collagen and other cuticle proteins in nematodes occursthrough di- and tri-tyrosine linkages which bridge and stabilize theproteinaceous structure (Fetterer et al., 1993; Fetterer and Rhoads,1990). Because peroxidases such as sea urchin ovoperoxidase and humanmyeloperoxidase carry out this reaction (Malanik and Ledvina, 1979;LaBella et al., 1968; Deits et al., 1984), we hypothesized that thefunction of Ce-Duox1 is to generate tyrosine cross-links, and that thedefective cuticle in the Ce-Duox RNAi animals is due to an inability toform tyrosine cross-links. A role for an unknown peroxidase in tyrosinecross-linking in Ascaris was previously suggested based on studies inwhich tyrosine cross-linking activity was inhibited using the peroxidaseinhibitors 4-amino-2,3,4 aminotriazole, phenylhydrazine, and N-acetyltyrosine (Fetterer et al., 1993). We therefore examined the wild-typeand Ce-Duox1 RNAi knockout animals for di- and tri-tyrosine linkages. AnHPLC profile of an acid hydrolysate of the wild-type C. elegans revealeda first large peak which was identified as dityrosine based oncomparison with authentic standard and mass spectral analysis, and thesecond peak is identified as trityrosine based on its migration on HPLCrelative to dityrosine and mass spectral analysis. Based on peak areasand assuming equivalent ionization, dityrosine and tyrosine were presentin a ratio of 1:200 in adult wild-type animals. In addition, thefluorescence excitation/emission maxima were determined at alkaline andacidic pH and were in good agreement with previously reported values(Jacob, et al. 1996). Neither the dityrosine nor the trityrosine peakswere detected in hydrolysates of Ce-Duox RNAi nematodes.

EXAMPLE 11

Participation of Duox in Cuticle Biogenesis, Ultrastructural Analysis

The similarity in phenotypes among animals defective in collagen andcuticle biosynthesis compared with the RNAi Duox animals suggested thatDuox participates in cuticle biogenesis. To confirm this hypothesis,electron microscopy was carried out on wild-type and RNAi animals.

Wild type or RNAi blistered adult C. elegans were collected and washedfirst with M9 buffer and then with 0.1 M cacodylate buffer (pH 7.4).Animals were pelleted, added to 1 ml of 0.8% glutaraldeyde, 0.7% osmiumtetroxide, 0.1 M cacodylate pH 7.4 and incubated on ice for 1.5 hourswith occasional mixing. The animals were washed with 0.1 M cacodylatebuffer, transferred to a glass depression slide and cut in half with a23 gauge needle. Bisected animals were transferred into a tubecontaining 1 ml of fresh fixative (0.8% glutaraldehyde, 0.7% osmiumtetroxide, 0.1 M cacodylate pH 7.4) and incubated on ice for 2 hrs.After washing with 0.1 M cacodylate buffer, the bisected animals werefixed overnight on ice in 1% osmium tetroxide in 0.1 M cacodylatebuffer. Animals were washed several times in 0.1 M cacodylate buffer,dehydrated using graded alcohols through propylene oxide, infiltratedand embedded in Embed-812 (Electron Microscopy Sciences, Ft. Washington,Pa.). The animals were teased into parallel arrangement with an eyelashprobe prior to polymerization at 60° C. for 16 hours. Sections (0.5 mm)were evaluated for orientation and ultrasections (800 Å thick) werecollected on 200 mesh copper grids, stained with uranyl acetate and leadcitrate, and cross sections were examined with a Philips EM201 electronmicroscope.

Ultrastructural analysis revealed that cuticle of RNAi Duox animals wasgrossly abnormal In normal animals three cuticle layers were seenclearly: the cortical (outer), median and basal (inner) layer, asdescribed previously (Cox, G. N., et al., 1981). The median layer iscomposed of struts connecting the cortical and basal layers, with afluid-filled space between these layers. The RNAi animals frequentlyshowed separation between the cortical and the basal layers, with markedexpansion of the fluid cavity and broken and distended struts that werestill visible on these layers. These separations occurred mainly overbundles of muscle fiber and are likely to account for the formation ofthe blisters observed using light microscopy. Thus, the cuticlestructure was severely affected in RNAi Duox animals.

EXAMPLE 12

Construction of Duox Peroxidase Domain Expression Plasmids

The polymerase chain reaction was used to amplify the peroxidase domainsof h-Duox (amino acid residues 1–593, SEQ ID NO:31) and Ce-Duox (aminoacid residues 1–590, SEQ ID NO:32) from the cloned full lengthsequences. The primers were designed to introduce an N-terminal BamH Isite and a C-terminal Not I site. PCR products were digested with BamH Iand Not I and ligated into the pET-32a(+) vector from Novogen (Madison,Wis.). Plasmids were transformed into BL21(DE3) cells containing thechloramphenicol-resistant plasmid pT-groE (Yasukawa, et al., 1995),which expresses the chaperonins groES and groEL from the T7 promoter.The pT-groE expression vector in BL21(DE3) cells was a generous giftfrom Dr. Lee-Ho Wang (University of Texas Health Science Center,Houston, Tex.) and Dr. Shunsuke Ishii (Institute of Physical andChemical Research, Ibaraki, Japan). LB-agar plates containing bothampicillin and chloramphenicol were used to isolate colonies.

Expression of Duox Peroxidase Domains

A 0.5 ml LB overnight culture of cells containing plasmid with theperoxidase domain from h-Duox or Ce-Duox was used to inoculate 50 ml ofmodified TB medium (Sandhu et al., 1993) containing 0.5 mMd-aminolevulinic acid, 100 mg/ml ampicillin and 25 mg/ml chloramphenicolin a 250 ml flask. Bacteria were grown at 37° C. in a shaker at 200 RPMuntil the cell density measured 0.7 OD at 600 nm.Isopropyl-b-D-thiogalactopyranoside (1 mM) was added and the culture wascontinued at 25° C. for 24 hours at 150 RPM. Cells were pelleted at4,500×g and resuspended in PBS containing4-(2-aminoethyl)benzenesulfonyl fluoride (2 nM), bestatin (130 nM),trans-epoxysuccinyl-L-leucyl-amido(4-guanidino)butane (1.4 nM) leupeptin(1 nM) and aprotinin (0.3 nM). The cell suspension was then sonicated onice.

Biochemical Activities of the Expressed Peroxidase Domains of Ce-Duox1and h-Duox1

The peroxidase domains of Ce-Duox (residues 1–590, SEQ ID NO:32) andh-Duox1 (residues 1–593, SEQ ID NO:31) were expressed in E. coli, asdescribed above. A lysate from these cells was analyzed for peroxidaseactivity. The results showed that the lysates from E. coli, expressingboth the human and the C. elegans peroxidase-homology domains from Duox,demonstrated peroxidase activity towards TMB, a well-characterizedperoxidase substrate. The activity was inhibited by the peroxidaseinhibitor aminobenzohydrazide. Lysates from E. coli expressing theperoxidase domains of h-Duox and Ce-Duox, but not those from vectorcontrol cells, also catalyzed the cross-linking of tyrosine ethyl ester.Two major fluorescent products were seen, as were also seen inhydrolysates of cuticle protein; peak 1 was identified byco-chromatography with authentic material and mass spectral analysis asdityrosine, while peak 2 was identified as tri-tyrosine by mass spectralanalysis as above.

EXAMPLE 13

Activity Assays

The 3,3′, 5,5′-tetramethylbenzidine (TMB) liquid substrate system(Sigma, St. Louis, Mo.) was used to assay peroxidase activity (Hollandet al., 1974). To 1 ml aliquots of the TMB substrate system 100 mg oflysate protein from cells expressing either the human Duox1 peroxidasedomain, Ce-Duox1 peroxidase domain or a vector control was added. Theperoxidase reactions were performed in triplicate and activity wasmonitored at 655 nm with a Beckman DU640B spectrophotometer. Somesamples contained 30 mM aminobenzoic acid hydrazide, a peroxidaseinhibitor (Kettle et al 1995).

To assay tyrosine cross-linking, tyrosine ethyl ester (20 mM) wasdissolved in 10 ml of PBS buffer supplemented with 80 ml of 3% H₂O₂. To1 ml aliquots, 100 mg of E. coli lysate protein was added, samples wereincubated for 1 hour, and the reaction was quenched using an equalvolume of 12 M HCl. Samples were analyzed for di- and tri-tyrosine asabove.

NADPH-Dependent Superoxide Generation Assay

In one embodiment of the present invention, NIH 3T3 cells stablytransfected with the human duox2 gene (SEQ ID NO:1) are analyzed forsuperoxide generation using the lucigenin (Bis-N-methylpyridiniumluminescence assay (Sigma, St. Louis, Mo., Li et al. (1998) J. Biol.Chem. 273, 2015–2023). Cells are washed with cold HANKS' solution andhomogenized on ice in HANKS' buffer containing 15% sucrose using aDounce homogenizer. Cell lysates are frozen immediately in a dryice/ethanol bath. For the assay, 30 μg of cell lysate is mixed with 200μM NADPH and 500 μM lucigenin. Luminescence is monitored using aLumiCounter (Packard) at three successive one minute intervals and thehighest value is used for comparison. Protein concentration isdetermined by the Bradford method.

Superoxide generation is monitored in lysates from some of the stablytransfected cell lines and was compared with superoxide generation bythe untransfected NIH 3T3 cell lysates. The results are show that thetransfected cells possess the highest degree of morphological changes bymicroscopic examination corresponding to the highest degree ofsuperoxide generation. The luminescent signal is inhibited by superoxidedismutase and the general flavoprotein inhibitor diphenylene iodonium,but is unaffected by added recombinant human p47phox, p67phox andRac1(GTP-γS), which are essential cytosolic factors for the phagocyterespiratory-burst oxidase.

In an alternate embodiment of the present invention, cells that arestably transfected with hduox2 (YA28) or with empty vector (NEF2) aregrown in 10 cm tissue culture plates in medium containing DMEM, 10% calfserum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 1 μg/mlpuromycin to approximately 80% confluency. Cells (five tissue cultureplates of each cell type) are washed briefly with 5 ml phosphatebuffered saline (PBS) then dissociated from the plates with PBScontaining 5 mM EDTA. Cells are pelleted by centrifuging briefly at1000×g.

To permeabilize the cells, freeze thaw lysis is carried out followed bypassage of the cell material through a small bore needle. Thesupernatant is removed and the cells frozen on dry ice for 15 minutes.After cells thaw, 200 μl lysis buffer (HANKS' Buffered SaltSolution—HBBS) containing a mixture of protease inhibitors from Sigma(Catalog #P2714) is added. Cells on ice are passed through an 18 guageneedle 10 times and 200 μl of HBSS buffer containing 34% sucrose wasadded to yield a final concentration of 17% sucrose. Sucrose appears toenhance stability upon storage. The combination of freeze-thawing andpassage through a needle results in lysis of essentially all of thecells, and this material is referred to as the “cell lysate.”

The cell lysates are assayed for protein concentration using the BioRadprotein assay system. Cell lysates are assayed for NADPH-dependentchemiluminescence by combining HBSS buffer, arachidonic acid, and 0.01–1μg protein in assay plates (96 well plastic plates). The reaction isinitiated by adding 1.5 mM NADPH and 75 μM lucigenin to the assay mix togive a final concentration of 200 μM NADPH and 10 μM lucigenin, and thechemiluminescence is monitored immediately. The final assay volume isabout 150 μl. The optimal arachidonic acid concentration is betweenabout 50–100 μM. A Packard Lumicount luminometer is used to measurechemiluminescence of the reaction between lucigenin and superoxide at37° C. The plate is monitored continuously for 60 minutes and themaximal relative luminescence unit (RLU) value for each sample isplotted. Results show that the presence of NaCl or KCl within aconcentration range of 50–150 μM is important for optimal activity.MgCl₂ (1–5 mM) further enhances activity by about 2-fold. This cell-freeassay for duox2 NADPH-oxidase activity is useful for screeningmodulators (inhibitors or stimulators) of the duox2 enzyme. The assaymay also be used to detect and duox NADPH-oxidase activity in generaland to screen for modulators (inhibitors or stimulators) of the duoxfamily of enzymes.

Nitro Blue Tetrazolium Reduction by Superoxide Generated by NIH 3T3Cells Transfected with the Duox2 cDNA (SEQ ID NO:1)

Superoxide generation by intact cells is monitored by using superoxidedismutase-sensitive reduction of nitroblue tetrazolium. NEF2 (vectoralone control), YA26 (duox2 (SEQ ID NO:1)-transfected) and YA28 (duox2(SEQ ID NO:1)-transfected) cells are plated in six well plates at500,000 cells per well. About 24 hours later, medium is removed fromcells and the cells are washed once with 1 mL Hanks solution (Sigma, St.Louis, Mo.). About 1 mL of filtered 0.25% Nitro blue tetrazolium (NBT,Sigma) is added in Hanks without or with 600 units of superoxidedismutase (Sigma) and cells are incubated at 37° C. in the presence of5% CO₂. After 8 minutes the cells are scraped and pelleted at more than10,000 g. The pellet is re-suspended in 1 mL of pyridine (Sigma) andheated for 10 minutes at 100° C. to solubilize the reduced NBT. Theconcentration of reduced NBT is determined by measuring the absorbanceat 510 nm, using an extinction coefficient of 11,000 M⁻¹ cm⁻¹. Somewells are untreated and used to determine cell number.

The data indicate that the duox2 (SEQ ID NO:1)-transfected cellsgenerated significant quantities of superoxide. Because superoxidedismutase is not likely to penetrate cells, superoxide must be generatedextracellularly. The amount of superoxide generated by these cells isabout 5–10% of that generated by activated human neutrophils.

EXAMPLE 14

Modification of Intracellular Components in Duox2 Transfected Cells

To test whether superoxide generated by duox2 can affect intracellular“targets”, aconitase activity in control and duox-transfected cell linesis monitored using methods as described in Suh et al. (1999) Nature 401,79–82. Aconitase contains a four-iron-sulphur cluster that is highlysusceptible to modification by superoxide, resulting in a loss ofactivity, and has been used as a reporter of intra-cellular superoxidegeneration. Aconitnase activity is determined as described in Gardner etal. (1995) J. Biol. Chem. 270, 13399–13405. Acotinase activity issignificantly diminished in all three duox-transfected cell linesdesignated YA26, YA28 and YA212 as compared to the transfected control.Approximately 50% of the aconitase in these cells is mitochondrial,based on differential centrifugation, and the cytosolic andmitochondrial forms are both affected. Control cytosolic andmitochondrial enzymes that do not contain iron-sulfur centres are notaffected. Superoxide generated in duox2-transfected cells is thereforecapable of reacting with and modifying intracellular components.

EXAMPLE 15

Tumor Generation in Nude Mice Receiving Cells Transfected with the HumanDuox2 cDNA (SEQ ID NO:1)

About 2×10⁶ NIH 3T3 cells (either hduox2-transfected with SEQ ID NO:1 orcells transfected using empty vector) are injected subdermally into thelateral aspect of the neck of 4–5 week old nude mice. Three to six miceare injected for each of three duox1-transfected cell lines, and 3 miceare injected with the cells transfected with empty vector (control).After 2 to 3 weeks, mice are sacrificed. The tumors are fixed in 10%formalin and characterized by histological analysis. Tumors averaged1.5×1×1 cm in size and show histology typical of sarcoma type tumors. Inaddition, tumors appear to be highly vascularized with superficialcapillaries. Eleven of twelve mice injected with duox2 gene-transfectedcells develop tumors, while none of the three control animals developtumors.

In another study, 15 mice are injected with duox2-transfected NIH 3T3cells. Of the 15 mice injected, 14 show large tumors within 17 days ofinjection, and tumors show expression of duox1 mRNA. Histologically, thetumors resemble fibrosarcomas and are similar to ras-induced tumors.Thus, ras and duox2 are similarly potent in their ability to inducetumorigenicity of NIH 3T3 cells in athymic mice.

EXAMPLE 16

Demonstration of the Role of Duox2 in Non-Cancerous Growth

A role in normal growth is demonstrated in rat aortic vascularsmooth-muscle cells by using antisense to rat duox2. Transfection withthe antisense DNA results in a decrease in both superoxide generationand serum-dependent growth. Duox2 is therefore implicated in normalgrowth in this cell type.

All patents, publications and abstracts cited above are incorporatedherein by reference in their entirety. It should be understood that theforegoing relates only to preferred embodiments of the present inventionand that numerous modifications or alterations may be made thereinwithout departing from the spirit and the scope of the present inventionas defined in the following claims.

1. An isolated protein capable of producing superoxide or havingperoxidase activity, wherein the protein comprises the amino acidsequence of SEQ ID NO:2, a fragment thereof, producing superoxide orhaving peroxidase activity or an addition thereto, a deletion thereof,or a conservative substitution thereof of no more than about 5% of theamino acid sequence, wherein the isolated protein is capable ofproducing superoxide, has peroxidase activity, reactions, or acombination thereof.
 2. The isolated protein of claim 1 wherein theconservative substitution comprises substitution of a) alanine, serine,or threonine for each other; b) aspartic acid or glutamic acid for eachother; c) asparagine or glutamine for each other; d) arginine or lysinefor each other; e) isoleucine, leucine, methionine, or valine for eachother; and f) phenylalanine, tyrosine, or tryptophan for each other. 3.An isolated protein comprising the amino acid sequence of SEQ ID NO:2 ora fragment thereof, wherein the isolated protein is capable of producingsuperoxide, has peroxidase activity, or a combination thereof.
 4. Anisolated nucleotide sequence encoding the protein, of claim
 1. 5. Anisolated nucleotide sequence encoding the protein of claim
 3. 6. Theisolated nucleotide sequence of claim 5, comprising SEQ ID NO:
 1. 7. Avector comprising the nucleotide sequence of claim
 5. 8. A vectorcomprising the nucleotide sequence of claim
 4. 9. An isolated host cellcomprising the vector of claim
 7. 10. An isolated host cell comprisingthe vector of claim
 8. 11. The isolated protein of claim 3, wherein theprotein comprises the amino acid sequence according to SEQ ID NO:2. 12.A vector comprising the nucleotide sequence of claim
 6. 13. An isolatedhost cell comprising the vector of claim
 12. 14. The isolated host cellof claim 13, wherein the host cell is a eukaryotic cell.
 15. Theisolated host cell of claim 13, wherein the host cell is a prokaryoticcell.
 16. The isolated protein of claim 3, wherein the protein comprisesamino acids 1283–1548 of SEQ LID NO:2.
 17. The isolated protein of claim3, wherein the protein comprises amino acids 1–593 of SEQ ID NO:2. 18.The isolated host cell of claim 9, wherein the host cell is a eukaryoticcell.
 19. The isolated host cell of claim 9, wherein the host cell is aprokaryotic cell.
 20. The isolated host cell of claim 10, wherein thehost cell is a eukaryotic cell.
 21. The isolated host cell of claim 10,wherein the host cell is a prokaryotic cell.